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Lu C, Jin A, Liu H, Gao C, Sun W, Zhang Y, Dai Q, Liu Y. Advancing tissue engineering through vascularized cell spheroids: building blocks of the future. Biomater Sci 2025; 13:1901-1922. [PMID: 40067332 DOI: 10.1039/d4bm01206b] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/09/2025]
Abstract
Vascularization is a crucial aspect of biofabrication, as the development of vascular networks is essential for tissue survival and the optimization of cellular functions. Spheroids have emerged as versatile units for vascularization, demonstrating significant potential in angiogenesis and prevascularization for tissue engineering and regenerative medicine. However, a major challenge in creating customized vascularized spheroids is the construction of a biomimetic extracellular matrix (ECM) microenvironment. This process requires careful regulation of environmental factors, including the modulation of growth factors, the selection of culture media, and the co-culture of diverse cell types. Recent advancements in biofabrication have expanded the potential applications of vascularized spheroids. The integration of microfluidic technology with bioprinting offers promising solutions to existing challenges in regenerative medicine. Spheroids have been widely studied for their ability to promote vascularization in in vitro models. This review highlights the latest developments in vascularized biofabrication, and systematically explores strategies for constructing vascularized spheroids. We provide a comprehensive analysis of spheroid applications in specific tissues, including skin, liver, bone, cardiac, and tumor models. Finally, the review addresses the major challenges and future directions in the field.
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Affiliation(s)
- Chunxiang Lu
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Aoxiang Jin
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Huazhen Liu
- School of Medicine, Shanghai University, Shanghai 200444, China
| | - Chuang Gao
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Wenbin Sun
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Yi Zhang
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Qiqi Dai
- School of Medicine, Shanghai University, Shanghai 200444, China
| | - Yuanyuan Liu
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
- National Center for Translational Medicine (Shanghai) SHU Branch, Shanghai, 200444, China
- Wenzhou Institute of Shanghai University, Wenzhou, 325000, China
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Spagnuolo FD, Kronemberger GS, Storey KJ, Kelly DJ. The maturation state and density of human cartilage microtissues influence their fusion and development into scaled-up grafts. Acta Biomater 2025; 194:109-121. [PMID: 39818242 DOI: 10.1016/j.actbio.2025.01.024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2024] [Revised: 12/09/2024] [Accepted: 01/13/2025] [Indexed: 01/18/2025]
Abstract
Functional cartilaginous tissues can potentially be engineered by bringing together numerous microtissues (µTs) and allowing them to fuse and re-organize into larger, structurally organized grafts. The maturation level of individual microtissues is known to influence their capacity to fuse, however its impact on the long-term development of the resulting tissue remains unclear. The first objective of this study was to investigate the influence of the maturation state of human bone-marrow mesenchymal stem/stromal cells (hBM-MSCSs) derived microtissues on their fusion capacity and the phenotype of the final engineered tissue. Less mature (day 2) cartilage microtissues were found to fuse faster, supporting the development of a matrix that was richer in sulphated glycosaminoglycans (sGAG) and collagen, while low in calcium deposits. This enhanced fusion in less mature microtissues correlated with enhanced expression of N-cadherin, followed by a progressive increase in markers associated with cell-extracellular matrix (ECM) interactions. We then engineered larger constructs with varying initial numbers (50, 150 or 300 µTs per well) of less mature microtissues, observing enhanced sGAG synthesis with increased microtissue density. We finally sought to engineer a scaled-up cartilage graft by fusing 4,000 microtissues and maintaining the resulting constructs under either dynamic or static culture conditions. Robust and reliable fusion was observed between microtissues at this scale, with no clear benefit of dynamic culture on the levels of matrix accumulation or the tensile modulus of the resulting construct. These results support the use of BM-MSCs derived microtissues for the development of large-scale, engineered functional cartilaginous grafts. STATEMENT OF SIGNIFICANCE: Microtissues are gaining attention for their use as biological building blocks in the field of tissue engineering. The fusion of multiple microtissues is crucial for achieving a cohesive engineered tissue of scale, however the impact of their maturation level on the long-term properties of the engineered graft is poorly understood. This paper emphasizes the importance of using less mature cartilage microtissues for supporting appropriate cell-cell interactions and robust chondrogenesis in vitro. We demonstrate that tissue development is not negatively impacted by increasing the initial numbers of microtissues within the graft. This biofabrication strategy has significant translation potential, as it enables the engineering of scaled-up cartilage grafts of clinically relevant sizes using bone marrow derived MSCs.
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Affiliation(s)
- Francesca D Spagnuolo
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; Department of Mechanical, Manufacturing and Biomedical Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland
| | - Gabriela S Kronemberger
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; Department of Mechanical, Manufacturing and Biomedical Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland
| | - Kyle J Storey
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; Department of Mechanical, Manufacturing and Biomedical Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland
| | - Daniel J Kelly
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; Department of Mechanical, Manufacturing and Biomedical Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland; Department of Anatomy and Regenerative Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin, Dublin, Ireland.
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Zhu T, Hu Y, Cui H, Cui H. 3D Multispheroid Assembly Strategies towards Tissue Engineering and Disease Modeling. Adv Healthc Mater 2024; 13:e2400957. [PMID: 38924326 DOI: 10.1002/adhm.202400957] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2024] [Revised: 06/21/2024] [Indexed: 06/28/2024]
Abstract
Cell spheroids (esp. organoids) as 3D culture platforms are popular models for representing cell-cell and cell-extracellular matrix (ECM) interactions, bridging the gap between 2D cell cultures and natural tissues. 3D cell models with spatially organized multiple cell types are preferred for gaining comprehensive insights into tissue pathophysiology and constructing in vitro tissues and disease models because of the complexities of natural tissues. In recent years, an assembly strategy using cell spheroids (or organoids) as living building blocks has been developed to construct complex 3D tissue models with spatial organization. Here, a comprehensive overview of recent advances in multispheroid assembly studies is provided. The different mechanisms of the multispheroid assembly techniques, i.e., automated directed assembly, noncontact remote assembly, and programmed self-assembly, are introduced. The processing steps, advantages, and technical limitations of the existing methodologies are summarized. Applications of the multispheroid assembly strategies in disease modeling, drug screening, tissue engineering, and organogenesis are reviewed. Finally, this review concludes by emphasizing persistent issues and future perspectives, encouraging researchers to adopt multispheroid assembly techniques for generating advanced 3D cell models that better resemble real tissues.
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Affiliation(s)
- Tong Zhu
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China
| | - Yan Hu
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China
| | - Haitao Cui
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China
| | - Haijun Cui
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China
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Baptista LS, Mironov V, Koudan E, Amorim ÉA, Pampolha TP, Kasyanov V, Kovalev A, Senatov F, Granjeiro JM. Bioprinting Using Organ Building Blocks: Spheroids, Organoids, and Assembloids. Tissue Eng Part A 2024; 30:377-386. [PMID: 38062998 DOI: 10.1089/ten.tea.2023.0198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2024] Open
Abstract
Three-dimensional (3D) bioprinting, a promising advancement in tissue engineering technology, involves the robotic, layer-by-layer additive biofabrication of functional 3D tissue and organ constructs. This process utilizes biomaterials, typically hydrogels and living cells, following digital models. Traditional tissue engineering uses a classic triad of living cells, scaffolds, and physicochemical signals in bioreactors. A scaffold is a temporary, often biodegradable, support structure. Tissue engineering primarily falls into two categories: (i) scaffold based and (ii) scaffold free. The latter, scaffold-free 3D bioprinting, is gaining increasing popularity. Organ building blocks (OBB), capable of self-assembly and self-organization, such as tissue spheroids, organoids, and assembloids, have begun to be utilized in scaffold-free bioprinting. This article discusses the expanding range of OBB, presents the rapidly evolving collection of bioprinting and bioassembly methods using these OBB, and finally, outlines the advantages, challenges, and future perspectives of using OBB in organ printing.
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Affiliation(s)
- Leandra Santos Baptista
- Campus Duque de Caxias Prof Geraldo Cidade, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Post-graduation Program in Biotechnology, National Institute of Metrology, Quality, and Technology (Inmetro), Rio de Janeiro, Brazil
- Laboratory of Eukaryotic Cell Biology, National Institute of Metrology, Quality and Technology (Inmetro), Rio de Janeiro, Brazil
| | - Vladimir Mironov
- Campus Duque de Caxias Prof Geraldo Cidade, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Elizaveta Koudan
- Center for Biomedical Engineering, National University of Science and Technology "MISIS," Moscow, Russia
| | - Érica Almeida Amorim
- Campus Duque de Caxias Prof Geraldo Cidade, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Gcell 3D, Rio de Janeiro, Brazil
- Precision Medicine Research Center, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Tathiana Proença Pampolha
- Post-graduation Program in Biotechnology, National Institute of Metrology, Quality, and Technology (Inmetro), Rio de Janeiro, Brazil
- Laboratory of Eukaryotic Cell Biology, National Institute of Metrology, Quality and Technology (Inmetro), Rio de Janeiro, Brazil
| | - Vladimir Kasyanov
- Joint Laboratory of Traumatology and Orthopaedics, Riga Stradins University, Riga, Latvia
| | - Alexei Kovalev
- Priorov Central National Institute of Traumatology and Orthopedics, Moscow, Russia
| | - Fedor Senatov
- Center for Biomedical Engineering, National University of Science and Technology "MISIS," Moscow, Russia
| | - José Mauro Granjeiro
- Post-graduation Program in Biotechnology, National Institute of Metrology, Quality, and Technology (Inmetro), Rio de Janeiro, Brazil
- Laboratory of Eukaryotic Cell Biology, National Institute of Metrology, Quality and Technology (Inmetro), Rio de Janeiro, Brazil
- Laboratory of Clinical Research in Odontology, Fluminense Federal University (UFF), Niterói, Brazil
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Minne M, Terrie L, Wüst R, Hasevoets S, Vanden Kerchove K, Nimako K, Lambrichts I, Thorrez L, Declercq H. Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering. Biofabrication 2024; 16:025035. [PMID: 38437715 DOI: 10.1088/1758-5090/ad2fd5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Accepted: 03/04/2024] [Indexed: 03/06/2024]
Abstract
Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such asin vitrodrug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool forin vitrodrug-testing and human disease modeling.
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Affiliation(s)
- Mendy Minne
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
| | - Lisanne Terrie
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
| | - Rebecca Wüst
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
| | - Steffie Hasevoets
- Biomedical Research Institute (BIOMED), Faculty of Medicine and Life Sciences, UHasselt, Diepenbeek, Belgium
| | - Kato Vanden Kerchove
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
| | - Kakra Nimako
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
| | - Ivo Lambrichts
- Biomedical Research Institute (BIOMED), Faculty of Medicine and Life Sciences, UHasselt, Diepenbeek, Belgium
| | - Lieven Thorrez
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
| | - Heidi Declercq
- Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven campus KULAK, Kortrijk, Belgium
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Byun H, Lee S, Shin H. Bioassembly of multicellular spheroids to mimic complex tissue structure using surface-modified magnetized nanofibers. Biofabrication 2024; 16:025006. [PMID: 38198701 DOI: 10.1088/1758-5090/ad1cf2] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Accepted: 01/10/2024] [Indexed: 01/12/2024]
Abstract
Advancements in biofabrication have led to major strides toward creating authentic organ models; however, replicating intricate organ structures without scaffolds remains challenging. In this study, we introduce a method utilizing surface-modifiable magnetic nanofibers to achieve precise control over spheroid functions and geometrical features, allowing the creation of multiple functional domains within a single microtissue. We generated magnetized nanofibers by electrospinning magnetic nanoparticles dispersed in poly-L-lactic acid solution. These fibers were then coated with polydopamine (PD) to enhance their biological functions, particularly reactive oxygen species (ROS) scavenging. These PD-coated magnetic fibers (PMFs) had magnetic-responsive properties when incorporated into human dermal fibroblast spheroids (0.019 ± 0.001 emu g-1). Furthermore, PMFs within the spheroids effectively regulated ROS levels by upregulating the expression of key anti-oxidative genes such assuperoxide dismutase-1(2.2 ± 0.1) andglutathione peroxidase-1(2.6 ± 0.1). By exploiting the magnetic responsiveness of spheroids, we were able to assemble them into various structures such as linear, triangular, and square structures using remotely applied magnetic forces. Within the assembled three-dimensional constructs, the cells in spheroids incorporating PMFs demonstrated resistance to ROS regulatory activity in the presence of hydrogen peroxide, while spheroids composed of bare fibers exhibited high ROS levels. Furthermore, we assembled spheroids containing fibroblasts and endothelial cells into complex tissue structures resembling vessels under magnetic manipulation. This innovative method holds tremendous promise for organ modeling and regenerative medicine due to the unprecedented control it allows in developing microtissues that closely emulate real organs.
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Affiliation(s)
- Hayeon Byun
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
- BK21 FOUR Education and Research Group for Biopharmaceutical Innovation Leader, Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Sangmin Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
- BK21 FOUR Education and Research Group for Biopharmaceutical Innovation Leader, Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Heungsoo Shin
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
- BK21 FOUR Education and Research Group for Biopharmaceutical Innovation Leader, Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea
- Institute of Nano Science and Technology, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
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Chae S, Lee H, Ryu D, Kim G. Macroscale pseudo-spheroids fabricated using methacrylated collagen-coated cells. Theranostics 2024; 14:924-939. [PMID: 38250048 PMCID: PMC10797297 DOI: 10.7150/thno.92193] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Accepted: 12/21/2023] [Indexed: 01/23/2024] Open
Abstract
Rationale: Cell spheroids have shown great promise as tools for creating effective three-dimensional (3D) tissue models, facilitating tissue reconstruction and organoid development, due to their high cell density and efficient cellular interactions. However, a significant challenge persists in creating large-scale tissue structures with a 3D geometrical architecture using spheroids, due to the continual condensation and reorganization of cells and their environments. Methods: The spherical cell aggregates (pseudo-cell spheroids) or macroscale cell aggregates were obtained by coating each adipose-derived stem cell (hASC) with methacrylated collagen (Col-Ma). Subsequently, the coated cells were printed into an alginate supporting bath and photocrosslinked through exposure to UV light. To assess the effectiveness of this procedure on regenerative potential, the generated cell aggregates were compared with conventional cell spheroids and bioprinted cell constructs using immunofluorescent staining and quantification of myogenic-related gene expressions. Moreover, the bioconstructs were implanted into a mouse model with volumetric muscle loss to further elucidate their regenerative and functional recovery properties. Results: The use of Col-Ma as a cell-coating material enables the rapid and physical aggregation of cells within several hours, regardless of the cell type. Furthermore, Col-Ma-coated cell aggregates can provide relatively lower hypoxic conditions than cell spheroids fabricated using the hanging drop method owing to the thin porous Col-Ma layer coated on the cells. In addition, the resulting structures maintain their geometrical architecture following cell fusion and possess the potential for efficient scale-up and 3D complex shape formation, making them more suitable for clinical applications than conventional cell spheroids. Finally, the feasibility of the Col-Ma-coated cylindrical human adipose-derived stem cells aggregates was assessed through implantation in a mouse volumetric muscle loss model, showing a significantly higher regenerative ability of muscle tissue than the normally bioprinted cell construct. Conclusion: Our newly proposed method has meaningful potential for various tissue engineering applications, supported by the improved cellular activities and efficient muscle regeneration observed in both in vitro and in vivo studies, and organ-chip models.
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Affiliation(s)
- SooJung Chae
- Department of Precision Medicine, Sungkyunkwan University School of Medicine (SKKU-SOM) Suwon 16419, Republic of Korea
| | - Hyeongjin Lee
- Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea
| | - Dongryeol Ryu
- Biomedical Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - GeunHyung Kim
- Department of Precision Medicine, Sungkyunkwan University School of Medicine (SKKU-SOM) Suwon 16419, Republic of Korea
- Institute of Quantum Biophysics, Department of Biophysics, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea
- Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, Suwon 16419, Republic of Korea
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Wu Z, Huang D, Wang J, Zhao Y, Sun W, Shen X. Engineering Heterogeneous Tumor Models for Biomedical Applications. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2304160. [PMID: 37946674 PMCID: PMC10767453 DOI: 10.1002/advs.202304160] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/22/2023] [Revised: 09/16/2023] [Indexed: 11/12/2023]
Abstract
Tumor tissue engineering holds great promise for replicating the physiological and behavioral characteristics of tumors in vitro. Advances in this field have led to new opportunities for studying the tumor microenvironment and exploring potential anti-cancer therapeutics. However, the main obstacle to the widespread adoption of tumor models is the poor understanding and insufficient reconstruction of tumor heterogeneity. In this review, the current progress of engineering heterogeneous tumor models is discussed. First, the major components of tumor heterogeneity are summarized, which encompasses various signaling pathways, cell proliferations, and spatial configurations. Then, contemporary approaches are elucidated in tumor engineering that are guided by fundamental principles of tumor biology, and the potential of a bottom-up approach in tumor engineering is highlighted. Additionally, the characterization approaches and biomedical applications of tumor models are discussed, emphasizing the significant role of engineered tumor models in scientific research and clinical trials. Lastly, the challenges of heterogeneous tumor models in promoting oncology research and tumor therapy are described and key directions for future research are provided.
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Affiliation(s)
- Zhuhao Wu
- Department of Rheumatology and ImmunologyNanjing Drum Tower HospitalSchool of Biological Science and Medical EngineeringSoutheast UniversityNanjing210096China
| | - Danqing Huang
- Department of Rheumatology and ImmunologyNanjing Drum Tower HospitalSchool of Biological Science and Medical EngineeringSoutheast UniversityNanjing210096China
| | - Jinglin Wang
- Department of Rheumatology and ImmunologyNanjing Drum Tower HospitalSchool of Biological Science and Medical EngineeringSoutheast UniversityNanjing210096China
| | - Yuanjin Zhao
- Department of Rheumatology and ImmunologyNanjing Drum Tower HospitalSchool of Biological Science and Medical EngineeringSoutheast UniversityNanjing210096China
- Department of Gastrointestinal SurgeryThe First Affiliated HospitalWenzhou Medical UniversityWenzhou325035China
| | - Weijian Sun
- Department of Gastrointestinal SurgeryThe Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical UniversityWenzhou325027China
| | - Xian Shen
- Department of Rheumatology and ImmunologyNanjing Drum Tower HospitalSchool of Biological Science and Medical EngineeringSoutheast UniversityNanjing210096China
- Department of Gastrointestinal SurgeryThe First Affiliated HospitalWenzhou Medical UniversityWenzhou325035China
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Faber L, Yau A, Chen Y. Translational biomaterials of four-dimensional bioprinting for tissue regeneration. Biofabrication 2023; 16:012001. [PMID: 37757814 PMCID: PMC10561158 DOI: 10.1088/1758-5090/acfdd0] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Revised: 09/16/2023] [Accepted: 09/27/2023] [Indexed: 09/29/2023]
Abstract
Bioprinting is an additive manufacturing technique that combines living cells, biomaterials, and biological molecules to develop biologically functional constructs. Three-dimensional (3D) bioprinting is commonly used as anin vitromodeling system and is a more accurate representation ofin vivoconditions in comparison to two-dimensional cell culture. Although 3D bioprinting has been utilized in various tissue engineering and clinical applications, it only takes into consideration the initial state of the printed scaffold or object. Four-dimensional (4D) bioprinting has emerged in recent years to incorporate the additional dimension of time within the printed 3D scaffolds. During the 4D bioprinting process, an external stimulus is exposed to the printed construct, which ultimately changes its shape or functionality. By studying how the structures and the embedded cells respond to various stimuli, researchers can gain a deeper understanding of the functionality of native tissues. This review paper will focus on the biomaterial breakthroughs in the newly advancing field of 4D bioprinting and their applications in tissue engineering and regeneration. In addition, the use of smart biomaterials and 4D printing mechanisms for tissue engineering applications is discussed to demonstrate potential insights for novel 4D bioprinting applications. To address the current challenges with this technology, we will conclude with future perspectives involving the incorporation of biological scaffolds and self-assembling nanomaterials in bioprinted tissue constructs.
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Affiliation(s)
- Leah Faber
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, United States of America
| | - Anne Yau
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, United States of America
| | - Yupeng Chen
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, United States of America
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Bahrami H, Sichetti F, Puppo E, Vettori L, Liu Chung Ming C, Perry S, Gentile C, Pietroni N. Physically-based simulation of elastic-plastic fusion of 3D bioprinted spheroids. Biofabrication 2023; 15:045021. [PMID: 37607551 DOI: 10.1088/1758-5090/acf2cb] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Accepted: 08/22/2023] [Indexed: 08/24/2023]
Abstract
Spheroids are microtissues containing cells organized in a spherical shape whose diameter is usually less than a millimetre. Depending on the properties of the environment they are placed in, some nearby spheroids spontaneously fuse and generate a tissue. Given their potential to mimic features typical of body parts and their ability to assemble by fusing in permissive hydrogels, they have been used as building blocks to 3D bioprint human tissue parts. Parameters controlling the shape and size of a bioprinted tissue using fusing spheroid cultures include cell composition, hydrogel properties, and their relative initial position. Hence, simulating, anticipating, and then controlling the spheroid fusion process is essential to control the shape and size of the bioprinted tissue. This study presents the first physically-based framework to simulate the fusion process of bioprinted spheroids. The simulation is based on elastic-plastic solid and fluid continuum mechanics models. Both models use the 'smoothed particle hydrodynamics' method, which is based on discretizing the continuous medium into a finite number of particles and solving the differential equations related to the physical properties (e.g. Navier-Stokes equation) using a smoothing kernel function. To further investigate the effects of such parameters on spheroid shape and geometry, we performed sensitivity and morphological analysis to validate our simulations within-vitrospheroids. Through ourin-silicosimulations by changing the aforementioned parameters, we show that the proposed models appropriately simulate the range of the elastic-plastic behaviours ofin-vitrofusing spheroids to generate tissues of desired shapes and sizes. Altogether, this study presented a physically-based simulation that can provide a framework for monitoring and controlling the geometrical shape of spheroids, directly impacting future research using spheroids for tissue bioprinting.
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Affiliation(s)
- Hassan Bahrami
- Faculty of Engineering and Information Technology, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
| | | | - Enrico Puppo
- Department of Computer Science, University of Genova, Genova, Italy
| | - Laura Vettori
- Faculty of Engineering and Information Technology, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
| | - Clara Liu Chung Ming
- Faculty of Engineering and Information Technology, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
| | - Stuart Perry
- Faculty of Engineering and Information Technology, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
| | - Carmine Gentile
- Faculty of Engineering and Information Technology, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
| | - Nico Pietroni
- Faculty of Engineering and Information Technology, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
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Pan R, Yang X, Ning K, Xie Y, Chen F, Yu L. Recapitulating the Drifting and Fusion of Two-Generation Spheroids on Concave Agarose Microwells. Int J Mol Sci 2023; 24:11967. [PMID: 37569343 PMCID: PMC10419262 DOI: 10.3390/ijms241511967] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2023] [Revised: 07/03/2023] [Accepted: 07/05/2023] [Indexed: 08/13/2023] Open
Abstract
Cells with various structures and proteins naturally come together to cooperate in vivo. This study used cell spheroids cultured in agarose micro-wells as a 3D model to study the movement of cells or spheroids toward other spheroids. The formation dynamics of tumor spheroids and the interactions of two batches of cells in the agarose micro-wells were studied. The results showed that a concave bottom micro-well (diameter: 2 mm, depth: 2 mm) prepared from 3% agarose could be used to study the interaction of two batches of cells. The initial tumor cell numbers from 5 × 103 cells/well to 6 × 104 cells/well all could form 3D spheroids after 3 days of incubation. Adding the second batch of DU 145 cells to the existing DU 145 spheroid resulted in the formation of satellite cell spheroids around the existing parental tumor spheroid. Complete fusion of two generation cell spheroids was observed when the parental spheroids were formed from 1 × 104 and 2 × 104 cells, and the second batch of cells was 5 × 103 per well. A higher amount of the second batch of cells (1 × 104 cell/well) led to the formation of independent satellite spheroids after 48 h of co-culture, suggesting the behavior of the second batch of cells towards existing parental spheroids depended on various factors, such as the volume of the parental spheroids and the number of the second batch cells. The interactions between the tumor spheroids and Human Umbilical Vein Endothelial Cells (HUVECs) were modeled on concave agarose micro-wells. The HUVECs (3 × 103 cell/well) were observed to gather around the parental tumor spheroids formed from 1 × 104, 2 × 104, and 3 × 104 cells per well rather than aggregate on their own to form HUVEC spheroids. This study highlights the importance of analyzing the biological properties of cells before designing experimental procedures for the sequential fusion of cell spheroids. The study further emphasizes the significant roles that cell density and the volume of the spheroids play in determining the location and movement of cells.
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Affiliation(s)
| | | | | | | | | | - Ling Yu
- Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, Institute for Clean Energy and Advanced Materials, School of Materials and Energy, Southwest University, Chongqing 400715, China; (R.P.); (X.Y.); (K.N.); (Y.X.); (F.C.)
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12
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Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids. Mater Today Bio 2023; 19:100603. [PMID: 37009070 PMCID: PMC10060184 DOI: 10.1016/j.mtbio.2023.100603] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Revised: 02/13/2023] [Accepted: 03/06/2023] [Indexed: 03/15/2023] Open
Abstract
The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.
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13
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Lee S, Choi S, Byun H, Lee J, Kwon H, Shin H. Composite Multicellular Spheroids Containing Fibers with Pores and Epigallocatechin Gallate (EGCG) Coating on the Surface for Enhanced Proliferation of Stem Cells. Macromol Biosci 2022; 22:e2200195. [PMID: 36111565 DOI: 10.1002/mabi.202200195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 08/23/2022] [Indexed: 01/15/2023]
Abstract
Multicellular spheroids are formed by strong cell-cell and cell-extracellular matrix interactions and are widely utilized in tissue engineering for therapeutic treatments or ex vivo tissue modeling. However, diffusion of oxygen into the spheroid gradually decreases, forming a necrotic core. In this study, polycaprolactone (PCL) fibers with pores and epigallocatechin gallate (EGCG) coating on their surface to provide a structural framework within the spheroids and investigated their ability to mitigate diffusional limitation and control over the proliferation of human adipose-derived stem cells (hADSCs) is engineered. The DNA content of composite spheroids prepared from fibers and hADSCs decreased in unadjusted cells (1224 ± 134 ng), in those with fibers with a smooth surface (SF) (1447 ± 331 ng), and in those EGCG-coated with SF (E-SF) (1437 ± 289 ng). Cells with fibers with pores on the surface (PF) (2020 ± 32 ng) and those with EGCG-coated PF (E-PF) (1911 ± 80 ng) increased after 7 days of culture, with a significantly greater number of proliferating cells (29 ± 8% and 30 ± 8%, respectively). These results indicate that physical modification through the formation of pores on the fiber surface alleviates diffusion limitation of composite spheroids, playing a dominant role over chemical modification.
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Affiliation(s)
- Sangmin Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea
| | - Soomi Choi
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea.,Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea
| | - Hayeon Byun
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea
| | - Jinkyu Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea
| | - Hyunseok Kwon
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea.,Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea
| | - Heungsoo Shin
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea.,Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea.,Institute of Nano Science and Technology, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea
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14
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Banerjee D, Singh YP, Datta P, Ozbolat V, O'Donnell A, Yeo M, Ozbolat IT. Strategies for 3D bioprinting of spheroids: A comprehensive review. Biomaterials 2022; 291:121881. [DOI: 10.1016/j.biomaterials.2022.121881] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Revised: 10/04/2022] [Accepted: 10/23/2022] [Indexed: 11/17/2022]
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15
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Correlation of the regenerative potential of dermal fibroblasts in 2D culture with the biological properties of fibroblast-derived tissue spheroids. Cell Tissue Res 2022; 390:453-464. [PMID: 36129531 DOI: 10.1007/s00441-022-03690-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 09/14/2022] [Indexed: 11/02/2022]
Abstract
In situ 3D bioprinting is a new emerging therapeutic modality for treating human skin diseases. The tissue spheroids have been previously suggested as a powerful tool in rapidly expanding bioprinting technology. It has been demonstrated that the regenerative potential of human dermal fibroblasts could be quantitatively evaluated in 2D cell culture and confirmed after implantation in vivo. However, the development of unbiassed quantitative criteria of the regenerative potential of 3D tissue spheroids in vitro before their in situ bioprinting remains to be investigated. Here it has been demonstrated for the first time that specific correlations exist between the regenerative potential of human dermal fibroblasts cultured in vitro as 2D cell monolayer with biological properties of 3D tissue spheroids fabricated from these fibroblasts. In vitro assessment of biological properties included diameter, spreading and fusion kinetics, and biomechanical properties of 3D tissue spheroids. This comprehensive characterization could be used to predict tissue spheroids' regenerative potential in vivo.
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16
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Deckers T, Hall GN, Papantoniou I, Aerts JM, Bloemen V. A platform for automated and label-free monitoring of morphological features and kinetics of spheroid fusion. Front Bioeng Biotechnol 2022; 10:946992. [PMID: 36091464 PMCID: PMC9461702 DOI: 10.3389/fbioe.2022.946992] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Accepted: 07/20/2022] [Indexed: 11/13/2022] Open
Abstract
Spheroids are widely applied as building blocks for biofabrication of living tissues, where they exhibit spontaneous fusion toward an integrated structure upon contact. Tissue fusion is a fundamental biological process, but due to a lack of automated monitoring systems, the in-depth characterization of this process is still limited. Therefore, a quantitative high-throughput platform was developed to semi-automatically select doublet candidates and automatically monitor their fusion kinetics. Spheroids with varying degrees of chondrogenic maturation (days 1, 7, 14, and 21) were produced from two different cell pools, and their fusion kinetics were analyzed via the following steps: (1) by applying a novel spheroid seeding approach, the background noise was decreased due to the removal of cell debris while a sufficient number of doublets were still generated. (2) The doublet candidates were semi-automatically selected, thereby reducing the time and effort spent on manual selection. This was achieved by automatic detection of the microwells and building a random forest classifier, obtaining average accuracies, sensitivities, and precisions ranging from 95.0% to 97.4%, from 51.5% to 92.0%, and from 66.7% to 83.9%, respectively. (3) A software tool was developed to automatically extract morphological features such as the doublet area, roundness, contact length, and intersphere angle. For all data sets, the segmentation procedure obtained average sensitivities and precisions ranging from 96.8% to 98.1% and from 97.7% to 98.8%, respectively. Moreover, the average relative errors for the doublet area and contact length ranged from 1.23% to 2.26% and from 2.30% to 4.66%, respectively, while the average absolute errors for the doublet roundness and intersphere angle ranged from 0.0083 to 0.0135 and from 10.70 to 13.44°, respectively. (4) The data of both cell pools were analyzed, and an exponential model was used to extract kinetic parameters from the time-series data of the doublet roundness. For both cell pools, the technology was able to characterize the fusion rate and quality in an automated manner and allowed us to demonstrate that an increased chondrogenic maturity was linked with a decreased fusion rate. The platform is also applicable to other spheroid types, enabling an increased understanding of tissue fusion. Finally, our approach to study spheroid fusion over time will aid in the design of controlled fabrication of “assembloids” and bottom-up biofabrication of living tissues using spheroids.
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Affiliation(s)
- Thomas Deckers
- Measure, Model and Manage Bioresponses (M3-BIORES), Department of Biosystems, KU Leuven, Leuven, Belgium
- Surface and Interface Engineered Materials (SIEM), Group T Leuven Campus, KU Leuven, Leuven, Belgium
- Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Leuven, Belgium
| | - Gabriella Nilsson Hall
- Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Leuven, Belgium
- Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium
| | - Ioannis Papantoniou
- Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Leuven, Belgium
- Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium
- Institute of Chemical Engineering Sciences, Foundation for Research and Technology—Hellas (FORTH), Patras, Greece
| | - Jean-Marie Aerts
- Measure, Model and Manage Bioresponses (M3-BIORES), Department of Biosystems, KU Leuven, Leuven, Belgium
- Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Leuven, Belgium
| | - Veerle Bloemen
- Surface and Interface Engineered Materials (SIEM), Group T Leuven Campus, KU Leuven, Leuven, Belgium
- Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Leuven, Belgium
- *Correspondence: Veerle Bloemen,
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17
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Arjoca S, Robu A, Neagu M, Neagu A. Mathematical and computational models in spheroid-based biofabrication. Acta Biomater 2022:S1742-7061(22)00418-4. [PMID: 35853599 DOI: 10.1016/j.actbio.2022.07.024] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 06/25/2022] [Accepted: 07/12/2022] [Indexed: 11/01/2022]
Abstract
Ubiquitous in embryonic development, tissue fusion is of interest to tissue engineers who use tissue spheroids or organoids as building blocks of three-dimensional (3D) multicellular constructs. This review presents mathematical models and computer simulations of the fusion of tissue spheroids. The motivation of this study stems from the need to predict the post-printing evolution of 3D bioprinted constructs. First, we provide a brief overview of differential adhesion, the main morphogenetic mechanism involved in post-printing structure formation. It will be shown that clusters of cohesive cells behave as an incompressible viscous fluid on the time scale of hours. The discussion turns then to mathematical models based on the continuum hydrodynamics of highly viscous liquids and on statistical mechanics. Next, we analyze the validity and practical use of computational models of multicellular self-assembly in live constructs created by tissue spheroid bioprinting. Finally, we discuss the perspectives of the field as machine learning starts to reshape experimental design, and modular robotic workstations tend to alleviate the burden of repetitive tasks in biofabrication. STATEMENT OF SIGNIFICANCE: Bioprinted constructs are living systems, which evolve via morphogenetic mechanisms known from developmental biology. This review presents mathematical and computational tools devised for modeling post-printing structure formation. They help achieving a desirable outcome without expensive optimization experiments. While previous reviews mainly focused on assumptions, technical details, strengths, and limitations of computational models of multicellular self-assembly, this article discusses their validity and practical use in biofabrication. It also presents an overview of mathematical models that proved to be useful in the evaluation of experimental data on tissue spheroid fusion, and in the calibration of computational models. Finally, the perspectives of the field are discussed in the advent of robotic biofabrication platforms and bioprinting process optimization by machine learning.
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Affiliation(s)
- Stelian Arjoca
- Center for Modeling Biological Systems and Data Analysis, Department of Functional Sciences, Victor Babes University of Medicine and Pharmacy Timisoara, Piata Eftimie Murgu Nr. 2-4, Timisoara 300041, Romania
| | - Andreea Robu
- Department of Automation and Applied Informatics, Politehnica University of Timisoara, Timisoara 300006, Romania
| | - Monica Neagu
- Center for Modeling Biological Systems and Data Analysis, Department of Functional Sciences, Victor Babes University of Medicine and Pharmacy Timisoara, Piata Eftimie Murgu Nr. 2-4, Timisoara 300041, Romania
| | - Adrian Neagu
- Center for Modeling Biological Systems and Data Analysis, Department of Functional Sciences, Victor Babes University of Medicine and Pharmacy Timisoara, Piata Eftimie Murgu Nr. 2-4, Timisoara 300041, Romania; Department of Physics & Astronomy, University of Missouri-Columbia, Columbia, MO 65211, USA.
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18
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Wolf KJ, Weiss JD, Uzel SGM, Skylar-Scott MA, Lewis JA. Biomanufacturing human tissues via organ building blocks. Cell Stem Cell 2022; 29:667-677. [PMID: 35523137 PMCID: PMC9617289 DOI: 10.1016/j.stem.2022.04.012] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The construction of human organs on demand remains a tantalizing vision to solve the organ donor shortage. Yet, engineering tissues that recapitulate the cellular and architectural complexity of native organs is a grand challenge. The use of organ building blocks (OBBs) composed of multicellular spheroids, organoids, and assembloids offers an important pathway for creating organ-specific tissues with the desired cellular-to-tissue-level organization. Here, we review the differentiation, maturation, and 3D assembly of OBBs into functional human tissues and, ultimately, organs for therapeutic repair and replacement. We also highlight future challenges and areas of opportunity for this nascent field.
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Affiliation(s)
- Kayla J Wolf
- Wyss Institute for Biologically Inspired Engineering & John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA
| | - Jonathan D Weiss
- Department of Bioengineering, Stanford University, 240 Pasteur Drive, Stanford, CA 94304, USA
| | - Sebastien G M Uzel
- Wyss Institute for Biologically Inspired Engineering & John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA
| | - Mark A Skylar-Scott
- Department of Bioengineering, Stanford University, 240 Pasteur Drive, Stanford, CA 94304, USA; BASE Initiative, Betty Irene Moore Children's Heart Center, Lucile Packard Children's Hospital, Stanford University School of Medicine, Stanford, CA 94304, USA.
| | - Jennifer A Lewis
- Wyss Institute for Biologically Inspired Engineering & John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA.
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19
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Beaune G, Sinkkonen L, Gonzalez-Rodriguez D, Timonen JVI, Brochard-Wyart F. Fusion Dynamics of Hybrid Cell-Microparticle Aggregates: A Jelly Pearl Model. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2022; 38:5296-5306. [PMID: 35109658 DOI: 10.1021/acs.langmuir.1c02949] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
We study the fusion of homogeneous cell aggregates and of hybrid aggregates combining cells and microparticles. In all cases, we find that the contact area does not vary linearly over time, as observed for liquid drops, but rather it follows a power law in t2/3. This result is interpreted by generalizing the fusion model of soft viscoelastic solid balls to viscoelastic liquid balls, akin to jelly pearls. We also explore the asymmetric fusion between a homogeneous aggregate and a hybrid aggregate. This latter experiment allows the determination of the self-diffusion coefficient of the cells in a tissue by following the spatial distribution of internalized particles in the cells.
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Affiliation(s)
- Grégory Beaune
- Department of Applied Physics, Aalto University School of Science, Puumiehenkuja 2, 02150 Espoo, Finland
| | - Laura Sinkkonen
- Department of Applied Physics, Aalto University School of Science, Puumiehenkuja 2, 02150 Espoo, Finland
| | | | - Jaakko V I Timonen
- Department of Applied Physics, Aalto University School of Science, Puumiehenkuja 2, 02150 Espoo, Finland
| | - Françoise Brochard-Wyart
- Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, 75005 Paris, France
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20
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Kim SJ, Byun H, Lee S, Kim E, Lee GM, Huh SJ, Joo J, Shin H. Spatially arranged encapsulation of stem cell spheroids within hydrogels for the regulation of spheroid fusion and cell migration. Acta Biomater 2022; 142:60-72. [PMID: 35085797 DOI: 10.1016/j.actbio.2022.01.047] [Citation(s) in RCA: 34] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Revised: 12/09/2021] [Accepted: 01/20/2022] [Indexed: 12/16/2022]
Abstract
Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity than individual cells in suspension. However, there is limited information on the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in a hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into a Matrigel for the investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in a Matrigel, denoted as sandwich culture, with a specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we performed a sandwich culture of human umbilical vein endothelial cells (HUVECs) and ASCs in co-cultured spheroids in the Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when the inter-spheroid distance was 200 µm. Similar results were obtained from an in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in a hydrogel. STATEMENT OF SIGNIFICANCE: Recently, spheroid-based 3D tissue culture techniques such as spheroid encapsulation or 3D printing are being intensively investigated for various purposes. However, there is limited research regarding the effect of the inter-spheroid distance on spheroid communication. Here, we demonstrate a spatially arranged spheroid encapsulation method within a Matrigel by using a temperature-responsive hydrogel. Human adipose-derived stem cell spheroids are encapsulated with a precisely controlled inter-spheroid distance from 100 to 400 µm and show different tendencies in cell migration and spheroid fusion. Our results suggest that the inter-spheroid distance affects spheroid communication, and thus, the inter-spheroid distance needs to be considered carefully according to the purpose.
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Affiliation(s)
- Se-Jeong Kim
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Hayeon Byun
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Sangmin Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Eunhyung Kim
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Gyeong Min Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Seung Jae Huh
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Jinmyoung Joo
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan 44919, Republic of Korea.
| | - Heungsoo Shin
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; BK21 FOUR, Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea; Institute of Nano Science and Technology (INST), Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea.
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21
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Kasamkattil J, Gryadunova A, Martin I, Barbero A, Schären S, Krupkova O, Mehrkens A. Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc. Int J Mol Sci 2022; 23:2530. [PMID: 35269672 PMCID: PMC8910276 DOI: 10.3390/ijms23052530] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 02/21/2022] [Accepted: 02/23/2022] [Indexed: 12/12/2022] Open
Abstract
Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.
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Affiliation(s)
- Jesil Kasamkattil
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
| | - Anna Gryadunova
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
- World-Class Research Center “Digital Biodesign and Personalized Healthcare”, Sechenov First Moscow State Medical University, 119435 Moscow, Russia
| | - Ivan Martin
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
| | - Andrea Barbero
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
| | - Stefan Schären
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
| | - Olga Krupkova
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
- Lepage Research Institute, University of Prešov, 17. Novembra 1, 081 16 Prešov, Slovakia
| | - Arne Mehrkens
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
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22
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Zhuang P, Chiang YH, Fernanda MS, He M. Using Spheroids as Building Blocks Towards 3D Bioprinting of Tumor Microenvironment. Int J Bioprint 2021; 7:444. [PMID: 34805601 PMCID: PMC8600307 DOI: 10.18063/ijb.v7i4.444] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Accepted: 10/02/2021] [Indexed: 12/12/2022] Open
Abstract
Cancer still ranks as a leading cause of mortality worldwide. Although considerable efforts have been dedicated to anticancer therapeutics, progress is still slow, partially due to the absence of robust prediction models. Multicellular tumor spheroids, as a major three-dimensional (3D) culture model exhibiting features of avascular tumors, gained great popularity in pathophysiological studies and high throughput drug screening. However, limited control over cellular and structural organization is still the key challenge in achieving in vivo like tissue microenvironment. 3D bioprinting has made great strides toward tissue/organ mimicry, due to its outstanding spatial control through combining both cells and materials, scalability, and reproducibility. Prospectively, harnessing the power from both 3D bioprinting and multicellular spheroids would likely generate more faithful tumor models and advance our understanding on the mechanism of tumor progression. In this review, the emerging concept on using spheroids as a building block in 3D bioprinting for tumor modeling is illustrated. We begin by describing the context of the tumor microenvironment, followed by an introduction of various methodologies for tumor spheroid formation, with their specific merits and drawbacks. Thereafter, we present an overview of existing 3D printed tumor models using spheroids as a focus. We provide a compilation of the contemporary literature sources and summarize the overall advancements in technology and possibilities of using spheroids as building blocks in 3D printed tissue modeling, with a particular emphasis on tumor models. Future outlooks about the wonderous advancements of integrated 3D spheroidal printing conclude this review.
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Affiliation(s)
- Pei Zhuang
- Department of Pharmaceutics, University of Florida, Gainesville, Florida, 32610, USA
| | - Yi-Hua Chiang
- Department of Pharmaceutics, University of Florida, Gainesville, Florida, 32610, USA
| | | | - Mei He
- Department of Pharmaceutics, University of Florida, Gainesville, Florida, 32610, USA
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23
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Rahimnejad M, Nasrollahi Boroujeni N, Jahangiri S, Rabiee N, Rabiee M, Makvandi P, Akhavan O, Varma RS. Prevascularized Micro-/Nano-Sized Spheroid/Bead Aggregates for Vascular Tissue Engineering. NANO-MICRO LETTERS 2021; 13:182. [PMID: 34409511 PMCID: PMC8374027 DOI: 10.1007/s40820-021-00697-1] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/26/2021] [Accepted: 07/13/2021] [Indexed: 05/02/2023]
Abstract
Efficient strategies to promote microvascularization in vascular tissue engineering, a central priority in regenerative medicine, are still scarce; nano- and micro-sized aggregates and spheres or beads harboring primitive microvascular beds are promising methods in vascular tissue engineering. Capillaries are the smallest type and in numerous blood vessels, which are distributed densely in cardiovascular system. To mimic this microvascular network, specific cell components and proangiogenic factors are required. Herein, advanced biofabrication methods in microvascular engineering, including extrusion-based and droplet-based bioprinting, Kenzan, and biogripper approaches, are deliberated with emphasis on the newest works in prevascular nano- and micro-sized aggregates and microspheres/microbeads.
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Affiliation(s)
- Maedeh Rahimnejad
- Biomedical Engineering Institute, School of Medicine, Université de Montréal, Montreal, Canada
- Research Centre, Centre Hospitalier de L'Université de Montréal (CRCHUM), Montreal, Canada
| | | | - Sepideh Jahangiri
- Research Centre, Centre Hospitalier de L'Université de Montréal (CRCHUM), Montreal, Canada
- Department of Biomedical Sciences, Faculty of Medicine, Université de Montréal, Montreal, Canada
| | - Navid Rabiee
- Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran, Iran.
| | - Mohammad Rabiee
- Biomaterial Group, Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran
| | - Pooyan Makvandi
- Centre for Materials Interfaces, Istituto Italiano Di Tecnologia, viale Rinaldo Piaggio 34, 56 025, Pontedera, Pisa, Italy
| | - Omid Akhavan
- Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran, Iran.
| | - Rajender S Varma
- Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute, Palacky University, Šlechtitelů 27, 783 71, Olomouc, Czech Republic.
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24
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Polonchuk L, Surija L, Lee MH, Sharma P, Liu Chung Ming C, Richter F, Ben-Sefer E, Rad MA, Mahmodi Sheikh Sarmast H, Shamery WA, Tran HA, Vettori L, Haeusermann F, Filipe EC, Rnjak-Kovacina J, Cox T, Tipper J, Kabakova I, Gentile C. Towards engineering heart tissues from bioprinted cardiac spheroids. Biofabrication 2021; 13. [PMID: 34265755 DOI: 10.1088/1758-5090/ac14ca] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Accepted: 07/15/2021] [Indexed: 02/06/2023]
Abstract
Currentin vivoandin vitromodels fail to accurately recapitulate the human heart microenvironment for biomedical applications. This study explores the use of cardiac spheroids (CSs) to biofabricate advancedin vitromodels of the human heart. CSs were created from human cardiac myocytes, fibroblasts and endothelial cells (ECs), mixed within optimal alginate/gelatin hydrogels and then bioprinted on a microelectrode plate for drug testing. Bioprinted CSs maintained their structure and viability for at least 30 d after printing. Vascular endothelial growth factor (VEGF) promoted EC branching from CSs within hydrogels. Alginate/gelatin-based hydrogels enabled spheroids fusion, which was further facilitated by addition of VEGF. Bioprinted CSs contracted spontaneously and under stimulation, allowing to record contractile and electrical signals on the microelectrode plates for industrial applications. Taken together, our findings indicate that bioprinted CSs can be used to biofabricate human heart tissues for long termin vitrotesting. This has the potential to be used to study biochemical, physiological and pharmacological features of human heart tissue.
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Affiliation(s)
- Liudmila Polonchuk
- F Hoffmann-La Roche AG Research and Development Division, Pharmaceutical Sciences, Roche Innovation Center Basel, Grenzacherstrasse 124, Basel, Basel-Stadt CH-4070, Switzerland
| | - Lydia Surija
- The University of Sydney Faculty of Medicine and Health, Kolling Building, Kolling Institute, St Leonards, Sydney, NSW 2065, Australia
| | - Min Ho Lee
- The University of Sydney Faculty of Medicine and Health, Kolling Building, Kolling Institute, St Leonards, Sydney, NSW 2065, Australia
| | - Poonam Sharma
- The University of Sydney Faculty of Medicine and Health, Kolling Building, Kolling Institute, St Leonards, Sydney, NSW 2065, Australia.,The University of Newcastle Faculty of Health and Medicine, University Drive, Callaghan, NSW 2308, Australia.,University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Clara Liu Chung Ming
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Florian Richter
- The University of Sydney Faculty of Medicine and Health, Kolling Building, Kolling Institute, St Leonards, Sydney, NSW 2065, Australia
| | - Eitan Ben-Sefer
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Maryam Alsadat Rad
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Hadi Mahmodi Sheikh Sarmast
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Wafa Al Shamery
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Hien A Tran
- School of Biomedical Engineering, UNSW Sydney, Sydney, NSW 2052, Australia
| | - Laura Vettori
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Fabian Haeusermann
- F Hoffmann-La Roche AG Research and Development Division, Pharmaceutical Sciences, Roche Innovation Center Basel, Grenzacherstrasse 124, Basel, Basel-Stadt CH-4070, Switzerland
| | - Elysse C Filipe
- Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia.,St Vincent Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2052, Australia
| | | | - Thomas Cox
- Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia.,St Vincent Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2052, Australia
| | - Joanne Tipper
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Irina Kabakova
- University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
| | - Carmine Gentile
- The University of Sydney Faculty of Medicine and Health, Kolling Building, Kolling Institute, St Leonards, Sydney, NSW 2065, Australia.,University of Technology Sydney Faculty of Engineering and IT, Building 11, Level 10, Room 115, Ultimo, Sydney, NSW 2007, Australia
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25
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Compera N, Atwell S, Wirth J, Wolfrum B, Meier M. Upscaling of pneumatic membrane valves for the integration of 3D cell cultures on chip. LAB ON A CHIP 2021; 21:2986-2996. [PMID: 34143169 PMCID: PMC8314520 DOI: 10.1039/d1lc00194a] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Accepted: 04/08/2021] [Indexed: 05/14/2023]
Abstract
Microfluidic large-scale integration (mLSI) technology enables the automation of two-dimensional (2D) cell culture processes in a highly parallel manner. Despite the wide range of biological applications of mLSI chips, manufacturing limitations of the central functional element, the pneumatic membrane valve (PMV), make the technology inaccessible for integrating tissue cultures and organoids with dimensions larger than tens of microns. In this study, we developed microtechnology processes to upscale PMVs for mLSI chips by combining 3D printing and soft lithography. Therefore, we developed a robust soft lithography protocol for the production of polydimethylsiloxane chips with PMVs from 3D-printed acrylate and wax molds. While scaled-up PMVs manufactured from acrylate-printed molds exhibited channel profiles with staircases, owing to the inherent 3D stereolithography printing process, PMVs manufactured from reflowed wax molds exhibited a semi-half-rounded channel profile. PMVs with different channel profiles showed closing pressures between 130 and 22.5 kPa, respectively. We demonstrated the functionality of the scaled-up PMVs by forming and maintaining 3D cell cultures from mouse fibroblasts (NIH3T3), human induced pluripotent stem cells (hiPSCs), and human adipose-derived adult stem cells (hASCs), with a narrow size distribution between 124 and 136 μm. Further, parallel and serial design of PMVs on an mLSI chip is used to first form and culture 3D cell cultures before fusing them within a defined flow process. Unit cell designs with upscaled PMVs enabled parallel formation, culturing, trapping, retrieval, and fusion of 3D cell cultures. Thus, the presented additive manufacturing strategy for mLSI chips will foster new developments for highly parallel 3D cell culture screening applications.
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Affiliation(s)
- Nina Compera
- Helmholtz Pioneer Campus, Helmholtz Zentrum München GmbH, German Research Center for Environmental Health, Munich, Germany.
| | - Scott Atwell
- Helmholtz Pioneer Campus, Helmholtz Zentrum München GmbH, German Research Center for Environmental Health, Munich, Germany.
| | - Johannes Wirth
- Helmholtz Pioneer Campus, Helmholtz Zentrum München GmbH, German Research Center for Environmental Health, Munich, Germany.
| | - Bernhard Wolfrum
- Neuroelectronics - Department of Electrical and Computer Engineering, Technical University of Munich, Germany
| | - Matthias Meier
- Helmholtz Pioneer Campus, Helmholtz Zentrum München GmbH, German Research Center for Environmental Health, Munich, Germany. and TUM School of Medicine, Technical University of Munich, Munich, Germany
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26
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Apelgren P, Amoroso M, Säljö K, Montelius M, Lindahl A, Stridh Orrhult L, Gatenholm P, Kölby L. Vascularization of tissue engineered cartilage - Sequential in vivo MRI display functional blood circulation. Biomaterials 2021; 276:121002. [PMID: 34274777 DOI: 10.1016/j.biomaterials.2021.121002] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 06/21/2021] [Accepted: 06/28/2021] [Indexed: 12/24/2022]
Abstract
Establishing functional circulation in bioengineered tissue after implantation is vital for the delivery of oxygen and nutrients to the cells. Native cartilage is avascular and thrives on diffusion, which in turn depends on proximity to circulation. Here, we investigate whether a gridded three-dimensional (3D) bioprinted construct would allow ingrowth of blood vessels and thus prove a functional concept for vascularization of bioengineered tissue. Twenty 10 × 10 × 3-mm 3Dbioprinted nanocellulose constructs containing human nasal chondrocytes or cell-free controls were subcutaneously implanted in 20 nude mice. Over the next 3 months, the mice were sequentially imaged with a 7 T small-animal MRI system, and the diffusion and perfusion parameters were analyzed. The chondrocytes survived and proliferated, and the shape of the constructs was well preserved. The diffusion coefficient was high and well preserved over time. The perfusion and diffusion patterns shown by MRI suggested that blood vessels develop over time in the 3D bioprinted constructs; the vessels were confirmed by histology and immunohistochemistry. We conclude that 3D bioprinted tissue with a gridded structure allows ingrowth of blood vessels and has the potential to be vascularized from the host. This is an essential step to take bioengineered tissue from the bench to clinical practice.
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Affiliation(s)
- Peter Apelgren
- Department of Plastic Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; Region Västra Götaland, Sahlgrenska University Hospital, Department of Plastic Surgery, Gothenburg, Sweden.
| | - Matteo Amoroso
- Department of Plastic Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; Region Västra Götaland, Sahlgrenska University Hospital, Department of Plastic Surgery, Gothenburg, Sweden
| | - Karin Säljö
- Department of Plastic Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; Region Västra Götaland, Sahlgrenska University Hospital, Department of Plastic Surgery, Gothenburg, Sweden
| | - Mikael Montelius
- Department of Radiation Physics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Anders Lindahl
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, Gothenburg, Sweden
| | - Linnea Stridh Orrhult
- 3D Bioprinting Centre, Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden
| | - Paul Gatenholm
- 3D Bioprinting Centre, Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden
| | - Lars Kölby
- Department of Plastic Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; Region Västra Götaland, Sahlgrenska University Hospital, Department of Plastic Surgery, Gothenburg, Sweden
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27
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Kronemberger GS, Beatrici A, Dalmônico GML, Rossi AL, Miranda GASC, Boldrini LC, Mauro Granjeiro J, Baptista LS. The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication. Artif Organs 2021; 45:1208-1218. [PMID: 34036603 DOI: 10.1111/aor.14000] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/05/2021] [Accepted: 05/18/2021] [Indexed: 11/30/2022]
Abstract
As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.
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Affiliation(s)
- Gabriela S Kronemberger
- Nucleus of Multidisciplinary Research in Biology (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil.,Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Post-graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Inmetro and Uezo, Duque de Caxias, Brazil
| | - Anderson Beatrici
- Post-graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Scientific and Technological Metrology Division (Dimci), National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil
| | | | | | - Guilherme A S C Miranda
- Nucleus of Multidisciplinary Research in Biology (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil.,Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Post-graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil
| | - Leonardo C Boldrini
- Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Post-graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Inmetro and Uezo, Duque de Caxias, Brazil.,Post-graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil
| | - José Mauro Granjeiro
- Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Post-graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Inmetro and Uezo, Duque de Caxias, Brazil.,Post-graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Laboratory of Clinical Research in Odontology, Fluminense Federal University (UFF), Niterói, Brazil
| | - Leandra Santos Baptista
- Nucleus of Multidisciplinary Research in Biology (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Xerém, Duque de Caxias, Brazil.,Laboratory of Tissue Bioengineering, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil.,Post-graduation Program of Translational Biomedicine (Biotrans), Unigranrio, Inmetro and Uezo, Duque de Caxias, Brazil.,Post-graduation Program in Biotechnology, National Institute of Metrology, Quality and Technology (Inmetro), Duque de Caxias, Brazil
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28
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Augustine R, Dan P, Hasan A, Khalaf IM, Prasad P, Ghosal K, Gentile C, McClements L, Maureira P. Stem cell-based approaches in cardiac tissue engineering: controlling the microenvironment for autologous cells. Biomed Pharmacother 2021; 138:111425. [PMID: 33756154 DOI: 10.1016/j.biopha.2021.111425] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 02/08/2021] [Accepted: 02/21/2021] [Indexed: 02/07/2023] Open
Abstract
Cardiovascular disease is one of the leading causes of mortality worldwide. Cardiac tissue engineering strategies focusing on biomaterial scaffolds incorporating cells and growth factors are emerging as highly promising for cardiac repair and regeneration. The use of stem cells within cardiac microengineered tissue constructs present an inherent ability to differentiate into cell types of the human heart. Stem cells derived from various tissues including bone marrow, dental pulp, adipose tissue and umbilical cord can be used for this purpose. Approaches ranging from stem cell injections, stem cell spheroids, cell encapsulation in a suitable hydrogel, use of prefabricated scaffold and bioprinting technology are at the forefront in the field of cardiac tissue engineering. The stem cell microenvironment plays a key role in the maintenance of stemness and/or differentiation into cardiac specific lineages. This review provides a detailed overview of the recent advances in microengineering of autologous stem cell-based tissue engineering platforms for the repair of damaged cardiac tissue. A particular emphasis is given to the roles played by the extracellular matrix (ECM) in regulating the physiological response of stem cells within cardiac tissue engineering platforms.
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Affiliation(s)
- Robin Augustine
- Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, 2713, Doha, Qatar; Biomedical Research Center (BRC), Qatar University, PO Box 2713, Doha, Qatar.
| | - Pan Dan
- Department of Cardiovascular and Transplantation Surgery, Regional Central Hospital of Nancy, Lorraine University, Nancy 54500, France; Department of Thoracic and Cardiovascular Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
| | - Anwarul Hasan
- Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, 2713, Doha, Qatar; Biomedical Research Center (BRC), Qatar University, PO Box 2713, Doha, Qatar.
| | | | - Parvathy Prasad
- International and Inter University Center for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, Kerala 686560, India
| | - Kajal Ghosal
- Dr. B. C. Roy College of Pharmacy and AHS, Durgapur 713206, India
| | - Carmine Gentile
- School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, NSW 2007, Australia; School of Medicine, Faculty of Medicine and Health, University of Sydney, NSW 2000, Australia; Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Lana McClements
- School of Life Sciences, Faculty of Science, University of Technology Sydney, NSW 2007, Australia
| | - Pablo Maureira
- Department of Cardiovascular and Transplantation Surgery, Regional Central Hospital of Nancy, Lorraine University, Nancy 54500, France
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29
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Roche CD, Sharma P, Ashton AW, Jackson C, Xue M, Gentile C. Printability, Durability, Contractility and Vascular Network Formation in 3D Bioprinted Cardiac Endothelial Cells Using Alginate-Gelatin Hydrogels. Front Bioeng Biotechnol 2021; 9:636257. [PMID: 33748085 PMCID: PMC7968457 DOI: 10.3389/fbioe.2021.636257] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Accepted: 02/01/2021] [Indexed: 12/30/2022] Open
Abstract
Background 3D bioprinting cardiac patches for epicardial transplantation are a promising approach for myocardial regeneration. Challenges remain such as quantifying printability, determining the ideal moment to transplant, and promoting vascularisation within bioprinted patches. We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural self-organisation into networks. Methods We evaluated 3D-bioprinted double-layer patches using alginate/gelatine (AlgGel) hydrogels and three extrusion bioprinters (REGEMAT3D, INVIVO, BIO X). Bioink contained either neonatal mouse cardiac cell spheroids or free (not-in-spheroid) human coronary artery endothelial cells with fibroblasts, mixed with AlgGel. To test the effects on durability, some patches were bioprinted as a single layer only, cultured under minimal movement conditions or had added fibroblast-derived extracellular matrix hydrogel (AlloECM). Controls included acellular AlgGel and gelatin methacryloyl (GELMA) patches. Results Printability was similar across bioprinters. For AlgGel compared to GELMA: resolutions were similar (200-700 μm line diameters), printing accuracy was 45 and 25%, respectively (AlgGel was 1.7x more accurate; p < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); p = 0.36. For durability, AlgGel patch median survival in culture was 14 days (IQR:10-27) overall which was not significantly affected by bioprinting system or cellular content in patches. We identified three factors which reduced durability in culture: (1) bioprinting one layer depth patches (instead of two layers); (2) movement disturbance to patches in media; and (3) the addition of AlloECM to AlgGel. Cells were viable after bioprinting followed by 28 days in culture, and all BIO X-bioprinted mouse cardiac cell spheroid patches presented contractile activity starting between day 7 and 13 after bioprinting. At day 28, endothelial cells in hydrogel displayed organisation into endothelial network-like structures. Conclusion AlgGel-based 3D bioprinted heart patches permit cardiomyocyte contractility and endothelial cell structural self-organisation. After bioprinting, a period of 2 weeks maturation in culture prior to transplantation may be optimal, allowing for a degree of tissue maturation but before many patches start to lose integrity. We quantify AlgGel printability and present novel factors which reduce AlgGel patch durability (layer number, movement, and the addition of AlloECM) and factors which had minimal effect on durability (bioprinting system and cellular patch content).
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Affiliation(s)
- Christopher David Roche
- Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.,School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, Sydney, NSW, Australia
| | - Poonam Sharma
- Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.,School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, Sydney, NSW, Australia.,Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia
| | - Anthony Wayne Ashton
- Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia
| | - Chris Jackson
- Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia
| | - Meilang Xue
- Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia
| | - Carmine Gentile
- Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.,School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, Sydney, NSW, Australia
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30
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3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels. Nat Commun 2021; 12:753. [PMID: 33531489 PMCID: PMC7854667 DOI: 10.1038/s41467-021-21029-2] [Citation(s) in RCA: 267] [Impact Index Per Article: 66.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 01/07/2021] [Indexed: 02/06/2023] Open
Abstract
Cellular models are needed to study human development and disease in vitro, and to screen drugs for toxicity and efficacy. Current approaches are limited in the engineering of functional tissue models with requisite cell densities and heterogeneity to appropriately model cell and tissue behaviors. Here, we develop a bioprinting approach to transfer spheroids into self-healing support hydrogels at high resolution, which enables their patterning and fusion into high-cell density microtissues of prescribed spatial organization. As an example application, we bioprint induced pluripotent stem cell-derived cardiac microtissue models with spatially controlled cardiomyocyte and fibroblast cell ratios to replicate the structural and functional features of scarred cardiac tissue that arise following myocardial infarction, including reduced contractility and irregular electrical activity. The bioprinted in vitro model is combined with functional readouts to probe how various pro-regenerative microRNA treatment regimes influence tissue regeneration and recovery of function as a result of cardiomyocyte proliferation. This method is useful for a range of biomedical applications, including the development of precision models to mimic diseases and the screening of drugs, particularly where high cell densities and heterogeneity are important.
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Daly AC, Prendergast ME, Hughes AJ, Burdick JA. Bioprinting for the Biologist. Cell 2021; 184:18-32. [PMID: 33417859 DOI: 10.1016/j.cell.2020.12.002] [Citation(s) in RCA: 158] [Impact Index Per Article: 39.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2020] [Revised: 10/29/2020] [Accepted: 12/01/2020] [Indexed: 12/30/2022]
Abstract
Building tissues from scratch to explore entirely new cell configurations could revolutionize fundamental understanding in biology. Bioprinting is an emerging technology to do this. Although typically applied to engineer tissues for therapeutic tissue repair or drug screening, there are many opportunities for bioprinting within biology, such as for exploring cellular crosstalk or cellular morphogenesis. The overall goals of this Primer are to provide an overview of bioprinting with the biologist in mind, outline the steps in extrusion bioprinting (the most widely used and accessible technology), and discuss alternative bioprinting technologies and future opportunities for bioprinting in biology.
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Affiliation(s)
- Andrew C Daly
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | | | - Alex J Hughes
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Jason A Burdick
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
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Gryadunova AA, Koudan EV, Rodionov SA, Pereira FDAS, Meteleva NY, Kasyanov VA, Parfenov VA, Kovalev AV, Khesuani YD, Mironov VA, Bulanova EA. Cytoskeleton systems contribute differently to the functional intrinsic properties of chondrospheres. Acta Biomater 2020; 118:141-152. [PMID: 33045401 DOI: 10.1016/j.actbio.2020.10.007] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 10/04/2020] [Accepted: 10/06/2020] [Indexed: 12/12/2022]
Abstract
Cytoskeleton systems, actin microfilaments, microtubules (MTs) and intermediate filaments (IFs) provide the biomechanical stability and spatial organization in cells. To understand the specific contributions of each cytoskeleton systems to intrinsic properties of spheroids, we've scrutinized the effects of the cytoskeleton perturbants, cytochalasin D (Cyto D), nocodazole (Noc) and withaferin A (WFA) on fusion, spreading on adhesive surface, morphology and biomechanics of chondrospheres (CSs). We confirmed that treatment with Cyto D but not with Noc or WFA severely affected CSs fusion and spreading dynamics and significantly reduced biomechanical properties of cell aggregates. Noc treatment affected spheroids spreading but not the fusion and surprisingly enhanced their stiffness. Vimentin intermediate filaments (VIFs) reorganization affected CSs spreading only. The analysis of all three cytoskeleton systems contribution to spheroids intrinsic properties was performed for the first time.
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Affiliation(s)
- Anna A Gryadunova
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation; Institute for Regenerative Medicine, I.M. Sechenov First Moscow State Medical University, Moscow 119991, Russian Federation.
| | - Elizaveta V Koudan
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation.
| | - Sergey A Rodionov
- N.N. Priorov National Medical Research Center of Traumatology and Orthopedics, Moscow 127299, Russian Federation
| | - F D A S Pereira
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation
| | - Nina Yu Meteleva
- I.D. Papanin Institute for Biology of Inland Waters RAS, Borok 152742, Russian Federation
| | - Vladimir A Kasyanov
- Riga Stradins University, Riga LV-1007, Latvia; Riga Technical University, Riga LV-1658, Latvia
| | - Vladislav A Parfenov
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation
| | - Alexey V Kovalev
- N.N. Priorov National Medical Research Center of Traumatology and Orthopedics, Moscow 127299, Russian Federation
| | - Yusef D Khesuani
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation
| | - Vladimir A Mironov
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation; Institute for Regenerative Medicine, I.M. Sechenov First Moscow State Medical University, Moscow 119991, Russian Federation
| | - Elena A Bulanova
- Laboratory for Biotechnological Research 3D Bioprinting Solutions, Moscow 115409, Russian Federation.
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Abstract
Microvasculature functions at the tissue and cell level, regulating local mass exchange of oxygen and nutrient-rich blood. While there has been considerable success in the biofabrication of large- and small-vessel replacements, functional microvasculature has been particularly challenging to engineer due to its size and complexity. Recently, three-dimensional bioprinting has expanded the possibilities of fabricating sophisticated microvascular systems by enabling precise spatiotemporal placement of cells and biomaterials based on computer-aided design. However, there are still significant challenges facing the development of printable biomaterials that promote robust formation and controlled 3D organization of microvascular networks. This review provides a thorough examination and critical evaluation of contemporary biomaterials and their specific roles in bioprinting microvasculature. We first provide an overview of bioprinting methods and techniques that enable the fabrication of microvessels. We then offer an in-depth critical analysis on the use of hydrogel bioinks for printing microvascularized constructs within the framework of current bioprinting modalities. We end with a review of recent applications of bioprinted microvasculature for disease modeling, drug testing, and tissue engineering, and conclude with an outlook on the challenges facing the evolution of biomaterials design for bioprinting microvasculature with physiological complexity.
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Affiliation(s)
- Ryan W. Barrs
- Bioengineering Department, Clemson University, Clemson, SC 29634, USA
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA
| | - Jia Jia
- Bioengineering Department, Clemson University, Clemson, SC 29634, USA
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA
| | - Sophia E. Silver
- Bioengineering Department, Clemson University, Clemson, SC 29634, USA
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA
| | - Michael Yost
- Department of Surgery, Medical University of South Carolina, Charleston, SC 29425, USA
| | - Ying Mei
- Bioengineering Department, Clemson University, Clemson, SC 29634, USA
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA
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Roche CD, Brereton RJL, Ashton AW, Jackson C, Gentile C. Current challenges in three-dimensional bioprinting heart tissues for cardiac surgery. Eur J Cardiothorac Surg 2020; 58:500-510. [PMID: 32391914 PMCID: PMC8456486 DOI: 10.1093/ejcts/ezaa093] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Revised: 01/27/2020] [Accepted: 02/18/2020] [Indexed: 12/25/2022] Open
Abstract
SUMMARY Previous attempts in cardiac bioengineering have failed to provide tissues for cardiac regeneration. Recent advances in 3-dimensional bioprinting technology using prevascularized myocardial microtissues as 'bioink' have provided a promising way forward. This review guides the reader to understand why myocardial tissue engineering is difficult to achieve and how revascularization and contractile function could be restored in 3-dimensional bioprinted heart tissue using patient-derived stem cells.
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Affiliation(s)
- Christopher D Roche
- Northern Clinical School of Medicine, University of Sydney, Kolling Institute, St Leonards, Sydney, NSW, Australia
- Department of Cardiothoracic Surgery, Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia
- Department of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney (UTS), Ultimo, Sydney, NSW, Australia
- Department of Cardiothoracic Surgery, University Hospital of Wales, Cardiff, UK
| | - Russell J L Brereton
- Department of Cardiothoracic Surgery, Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia
| | - Anthony W Ashton
- Northern Clinical School of Medicine, University of Sydney, Kolling Institute, St Leonards, Sydney, NSW, Australia
| | - Christopher Jackson
- Northern Clinical School of Medicine, University of Sydney, Kolling Institute, St Leonards, Sydney, NSW, Australia
| | - Carmine Gentile
- Northern Clinical School of Medicine, University of Sydney, Kolling Institute, St Leonards, Sydney, NSW, Australia
- Department of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney (UTS), Ultimo, Sydney, NSW, Australia
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Kosheleva NV, Efremov YM, Shavkuta BS, Zurina IM, Zhang D, Zhang Y, Minaev NV, Gorkun AA, Wei S, Shpichka AI, Saburina IN, Timashev PS. Cell spheroid fusion: beyond liquid drops model. Sci Rep 2020; 10:12614. [PMID: 32724115 PMCID: PMC7387529 DOI: 10.1038/s41598-020-69540-8] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Accepted: 05/19/2020] [Indexed: 01/14/2023] Open
Abstract
Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates-spheroids-have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation.
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Affiliation(s)
- Nastasia V Kosheleva
- FSBSI "Institute of General Pathology and Pathophysiology", 8, Baltiyskaya st., Moscow, 125315, Russia.
- FSBEI FPE "Russian Medical Academy of Continuous Professional Education" of the Ministry of Healthcare of Russia, 2/1, Barrikadnaya St., Moscow, 125993, Russia.
- Faculty of Biology, Lomonosov Moscow State University, 12-1, Leninskie Gory, Moscow, 119234, Russia.
| | - Yuri M Efremov
- Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2, Trubetskaya St., Moscow, 119991, Russia
| | - Boris S Shavkuta
- Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2, Trubetskaya St., Moscow, 119991, Russia
- Institute of Photonic Technologies, Research Center "Crystallography and Photonics" RAS, 2, Pionerskaya st., Troitsk, Moscow, 142190, Russia
| | - Irina M Zurina
- FSBSI "Institute of General Pathology and Pathophysiology", 8, Baltiyskaya st., Moscow, 125315, Russia
- FSBEI FPE "Russian Medical Academy of Continuous Professional Education" of the Ministry of Healthcare of Russia, 2/1, Barrikadnaya St., Moscow, 125993, Russia
- Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2, Trubetskaya St., Moscow, 119991, Russia
| | - Deying Zhang
- Department of Urology, Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| | - Yuanyuan Zhang
- Wake Forest University Institute for Regenerative Medicine, Winston-Salem, NC, USA
| | - Nikita V Minaev
- Institute of Photonic Technologies, Research Center "Crystallography and Photonics" RAS, 2, Pionerskaya st., Troitsk, Moscow, 142190, Russia
| | - Anastasiya A Gorkun
- FSBSI "Institute of General Pathology and Pathophysiology", 8, Baltiyskaya st., Moscow, 125315, Russia
- FSBEI FPE "Russian Medical Academy of Continuous Professional Education" of the Ministry of Healthcare of Russia, 2/1, Barrikadnaya St., Moscow, 125993, Russia
- Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2, Trubetskaya St., Moscow, 119991, Russia
| | - Shicheng Wei
- Department of Oral and Maxillofacial Surgery/Central Laboratory, Peking University School and Hospital of Stomatology, Beijing, 100081, China
- Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China
| | - Anastasia I Shpichka
- Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2, Trubetskaya St., Moscow, 119991, Russia
| | - Irina N Saburina
- FSBSI "Institute of General Pathology and Pathophysiology", 8, Baltiyskaya st., Moscow, 125315, Russia
- FSBEI FPE "Russian Medical Academy of Continuous Professional Education" of the Ministry of Healthcare of Russia, 2/1, Barrikadnaya St., Moscow, 125993, Russia
| | - Peter S Timashev
- Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2, Trubetskaya St., Moscow, 119991, Russia
- Institute of Photonic Technologies, Research Center "Crystallography and Photonics" RAS, 2, Pionerskaya st., Troitsk, Moscow, 142190, Russia
- Department of Polymers and Composites, N.N. Semenov Institute of Chemical Physics, 4, Kosygin st., Moscow, 119991, Russia
- Chemistry Department, Lomonosov Moscow State University, 1‑3, Leninskiye Gory, Moscow, 119991, Russia
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Turnbull G, Clarke J, Picard F, Zhang W, Riches P, Li B, Shu W. 3D biofabrication for soft tissue and cartilage engineering. Med Eng Phys 2020; 82:13-39. [PMID: 32709263 DOI: 10.1016/j.medengphy.2020.06.003] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2019] [Revised: 05/25/2020] [Accepted: 06/08/2020] [Indexed: 02/07/2023]
Abstract
Soft tissue injuries (STIs) affect patients of all age groups and represent a common worldwide clinical problem, resulting from conditions including trauma, infection, cancer and burns. Within the spectrum of STIs a mixture of tissues can be injured, ranging from skin to underlying nerves, blood vessels, tendons and cartilaginous tissues. However, significant limitations affect current treatment options and clinical demand for soft tissue and cartilage regenerative therapies continues to rise. Improving the regeneration of soft tissues has therefore become a key area of focus within tissue engineering. As an emerging technology, 3D bioprinting can be used to build complex soft tissue constructs "from the bottom up," by depositing cells, growth factors, extracellular matrices and other biomaterials in a layer-by-layer fashion. In this way, regeneration of cartilage, skin, vasculature, nerves, tendons and other bodily tissues can be performed in a patient specific manner. This review will focus on recent use of 3D bioprinting and other biofabrication strategies in soft tissue repair and regeneration. Biofabrication of a variety of soft tissue types will be reviewed following an overview of available cell sources, bioinks and bioprinting techniques.
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Affiliation(s)
- Gareth Turnbull
- Department of Biomedical Engineering, Wolfson Building, University of Strathclyde, 106 Rottenrow, Glasgow G4 0NW, United Kingdom; Department of Orthopaedic Surgery, Golden Jubilee National Hospital, Agamemnon St, Clydebank G81 4DY, United Kingdom
| | - Jon Clarke
- Department of Orthopaedic Surgery, Golden Jubilee National Hospital, Agamemnon St, Clydebank G81 4DY, United Kingdom
| | - Frédéric Picard
- Department of Biomedical Engineering, Wolfson Building, University of Strathclyde, 106 Rottenrow, Glasgow G4 0NW, United Kingdom; Department of Orthopaedic Surgery, Golden Jubilee National Hospital, Agamemnon St, Clydebank G81 4DY, United Kingdom
| | - Weidong Zhang
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Orthopedic Institute, Soochow University, Suzhou, Jiangsu, China
| | - Philip Riches
- Department of Biomedical Engineering, Wolfson Building, University of Strathclyde, 106 Rottenrow, Glasgow G4 0NW, United Kingdom
| | - Bin Li
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Orthopedic Institute, Soochow University, Suzhou, Jiangsu, China
| | - Wenmiao Shu
- Department of Biomedical Engineering, Wolfson Building, University of Strathclyde, 106 Rottenrow, Glasgow G4 0NW, United Kingdom.
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Pakhomova C, Popov D, Maltsev E, Akhatov I, Pasko A. Software for Bioprinting. Int J Bioprint 2020; 6:279. [PMID: 33088988 PMCID: PMC7557344 DOI: 10.18063/ijb.v6i3.279] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2020] [Accepted: 04/24/2020] [Indexed: 02/07/2023] Open
Abstract
The bioprinting of heterogeneous organs is a crucial issue. To reach the complexity of such organs, there is a need for highly specialized software that will meet all requirements such as accuracy, complexity, and others. The primary objective of this review is to consider various software tools that are used in bioprinting and to reveal their capabilities. The sub-objective was to consider different approaches for the model creation using these software tools. Related articles on this topic were analyzed. Software tools are classified based on control tools, general computer-aided design (CAD) tools, tools to convert medical data to CAD formats, and a few highly specialized research-project tools. Different geometry representations are considered, and their advantages and disadvantages are considered applicable to heterogeneous volume modeling and bioprinting. The primary factor for the analysis is suitability of the software for heterogeneous volume modeling and bioprinting or multimaterial three-dimensional printing due to the commonality of these technologies. A shortage of specialized suitable software tools is revealed. There is a need to develop a new application area such as computer science for bioprinting which can contribute significantly in future research work.
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Affiliation(s)
- Catherine Pakhomova
- Center for Design, Manufacturing and Materials, Skolkovo Institute of Science and Technology, Moscow.,Institute of Engineering Physics for Biomedicine, NRNU Mephi, Moscow
| | - Dmitry Popov
- Center for Design, Manufacturing and Materials, Skolkovo Institute of Science and Technology, Moscow
| | - Eugenii Maltsev
- Center for Design, Manufacturing and Materials, Skolkovo Institute of Science and Technology, Moscow
| | - Iskander Akhatov
- Center for Design, Manufacturing and Materials, Skolkovo Institute of Science and Technology, Moscow
| | - Alexander Pasko
- Center for Design, Manufacturing and Materials, Skolkovo Institute of Science and Technology, Moscow.,The National Centre for Computer Animation, Bournemouth University, UK
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Manning KL, Feder J, Kanellias M, Murphy J, Morgan JR. Toward Automated Additive Manufacturing of Living Bio-Tubes Using Ring-Shaped Building Units. SLAS Technol 2020; 25:608-620. [PMID: 32452278 DOI: 10.1177/2472630320920896] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Tissue engineering has been largely confined to academic research institutions with limited success in commercial settings. To help address this issue, more work is needed to develop new automated manufacturing processes for tissue-related technologies. In this article, we describe the automation of the funnel-guide, an additive manufacturing method that uses living tissue rings as building units to form bio-tubes. We developed a method based on 96-well plates and a modified off-the-shelf liquid-handling robot to retrieve, perform real-time quality control, and transfer tissue rings to the funnel-guide. Cells seeded into 96-well plates containing specially designed agarose micromolds self-assembled and formed ring-shaped microtissues that could be retrieved using a liquid-handling robot. We characterized the effects of time, cell type, and mold geometry on the morphology of the ring-shaped microtissues to inform optimal use of the building parts. We programmed and modified an off-the-shelf liquid-handling robot to retrieve ring-shaped microtissues from the 96-well plates, and we fabricated a custom illuminated pipette to visualize each ring-shaped microtissue prior to deposit in the funnel guide. Imaging at the liquid-air interface presented challenges that were overcome by controlling lighting conditions and liquid curvature. Based on these images, we incorporated into our workflow a real-time quality control step based on visual inspection and morphological criteria to assess each ring prior to use. We used this system to fabricate bio-tubes of endothelial cells with luminal alignment.
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Affiliation(s)
- Kali L Manning
- Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA
- Center for Biomedical Engineering, Brown University, Providence, RI, USA
| | - Jacob Feder
- Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA
| | - Marianne Kanellias
- Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA
- Center for Biomedical Engineering, Brown University, Providence, RI, USA
| | - John Murphy
- Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA
| | - Jeffrey R Morgan
- Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA
- Center for Biomedical Engineering, Brown University, Providence, RI, USA
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Injectable Therapeutic Organoids Using Sacrificial Hydrogels. iScience 2020; 23:101052. [PMID: 32353766 PMCID: PMC7191221 DOI: 10.1016/j.isci.2020.101052] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2020] [Revised: 03/11/2020] [Accepted: 04/03/2020] [Indexed: 12/28/2022] Open
Abstract
Organoids are becoming widespread in drug-screening technologies but have been used sparingly for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility in size and cellular organization. We introduce a method of using hydrogels as sacrificial scaffolds, which allow cells to form self-organized clusters followed by gentle release, resulting in highly reproducible multicellular structures on a large scale. We demonstrated this strategy for endothelial cells and mesenchymal stem cells to self-organize into blood-vessel units, which were injected into mice, and rapidly formed perfusing vasculature. Moreover, in a mouse model of peripheral artery disease, intramuscular injections of blood-vessel units resulted in rapid restoration of vascular perfusion within seven days. As cell therapy transforms into a new class of therapeutic modality, this simple method—by making use of the dynamic nature of hydrogels—could offer high yields of self-organized multicellular aggregates with reproducible sizes and cellular architectures.
Therapeutic, prevascularized organoids were formed in a sacrificial scaffold The organoids are highly reproducible and grown in a high-throughput manner The organoids rapidly formed perfusing vasculature in healthy mice Therapeutic potential was assessed in a mouse model of peripheral artery disease
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Kronemberger GS, Matsui RAM, Miranda GDASDCE, Granjeiro JM, Baptista LS. Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks. World J Stem Cells 2020; 12:110-122. [PMID: 32184936 PMCID: PMC7062040 DOI: 10.4252/wjsc.v12.i2.110] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2019] [Revised: 10/19/2019] [Accepted: 01/15/2020] [Indexed: 02/06/2023] Open
Abstract
Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating, in vitro, tissues with more authentic properties. Cell clusters called spheroids are the basis for scaffold-free tissue engineering. In this review, we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues. Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis, and are capable of spontaneously fusing to other spheroids, making them ideal building blocks for bone and cartilage tissue engineering. Here, we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro. Overall, recent studies support the notion that spheroids are ideal "building blocks" for tissue engineering by “bottom-up” approaches, which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting. Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.
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Affiliation(s)
- Gabriela S Kronemberger
- Laboratory of Tissue Bioengineering, Directory of Metrology Applied to Life Sciences, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Post-graduate Program in Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, RJ 25250-020, Brazil
| | - Renata Akemi Morais Matsui
- Laboratory of Tissue Bioengineering, Directory of Metrology Applied to Life Sciences, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Post-graduate Program in Biotechnology, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
| | - Guilherme de Almeida Santos de Castro e Miranda
- Laboratory of Tissue Bioengineering, Directory of Metrology Applied to Life Sciences, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Federal University of Rio de Janeiro (UFRJ), Campus Duque de Caxias, Duque de Caxias, RJ 25250-020, Brazil
| | - José Mauro Granjeiro
- Laboratory of Tissue Bioengineering, Directory of Metrology Applied to Life Sciences, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Post-graduate Program in Biotechnology, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Laboratory of Clinical Research in Odontology, Fluminense Federal University (UFF), Niterói 25255-030 Brazil
| | - Leandra Santos Baptista
- Laboratory of Tissue Bioengineering, Directory of Metrology Applied to Life Sciences, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Post-graduate Program in Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, RJ 25250-020, Brazil
- Post-graduate Program in Biotechnology, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Campus Duque de Caxias, Duque de Caxias, RJ 25245-390, Brazil
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Koudan EV, Gryadunova AA, Karalkin PA, Korneva JV, Meteleva NY, Babichenko II, Volkov AV, Rodionov SA, Parfenov VA, Pereira FDAS, Khesuani YD, Mironov VA, Bulanova EA. Multiparametric Analysis of Tissue Spheroids Fabricated from Different Types of Cells. Biotechnol J 2020; 15:e1900217. [DOI: 10.1002/biot.201900217] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2019] [Revised: 01/17/2020] [Indexed: 12/13/2022]
Affiliation(s)
- Elizaveta V. Koudan
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
| | - Anna A. Gryadunova
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
- Institute for Regenerative MedicineI. M. Sechenov First Moscow State Medical University Moscow 119991 Russia
| | - Pavel A. Karalkin
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
- Medical Research Radiological Centre of the Ministry of Health of the Russian Federation Moscow 125284 Russia
| | - Janetta V. Korneva
- I. D. Papanin Institute for Biology of Inland Waters RAS Borok 152742 Russia
| | - Nina Y. Meteleva
- I. D. Papanin Institute for Biology of Inland Waters RAS Borok 152742 Russia
| | - Igor I. Babichenko
- Peoples’ Friendship University of Russia (RUDN University) Moscow 117198 Russia
| | - Aleksey V. Volkov
- Peoples’ Friendship University of Russia (RUDN University) Moscow 117198 Russia
- N. N. Priorov National Medical Research Center of Traumatology and Orthopedics Moscow Russia
| | - Sergey A. Rodionov
- N. N. Priorov National Medical Research Center of Traumatology and Orthopedics Moscow Russia
| | - Vladislav A. Parfenov
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
| | - Frederico D. A. S. Pereira
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
| | - Yusef D. Khesuani
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
| | - Vladimir A. Mironov
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
- Institute for Regenerative MedicineI. M. Sechenov First Moscow State Medical University Moscow 119991 Russia
| | - Elena A. Bulanova
- Laboratory for Biotechnological Research 3D Bioprinting Solutions Kashirskoe Highway, 68‐2 Moscow 115409 Russia
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Kim EM, Lee YB, Kim SJ, Park J, Lee J, Kim SW, Park H, Shin H. Fabrication of core-shell spheroids as building blocks for engineering 3D complex vascularized tissue. Acta Biomater 2019; 100:158-172. [PMID: 31542503 DOI: 10.1016/j.actbio.2019.09.028] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2019] [Revised: 09/06/2019] [Accepted: 09/17/2019] [Indexed: 12/12/2022]
Abstract
Cell spheroids as building blocks for engineering micro-tissue should be able to mimic the complex structure of natural tissue. However, control of the distribution of multiple cell populations within cell spheroids is difficult to achieve with current spheroid-harvest methods such as hanging-drop and with the use of microwell plates. In this study, we report the fabrication of core-shell spheroids with the ultimate goal to form 3D complex micro-tissue. We used endothelial cells and two types of stem cells (human turbinate mesenchymal stem cells (hTMSCs)/adipose-derived stem cells (ADSCs)). The stem cells and endothelial cells formed layered micro-sized cell sheets (µCSs) on polydopamine micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids by expansion of the hydrogels. The co-cultured spheroids formed a core-shell structure irrespective of stem cell type. In addition, the size of the core-shell spheroids was controlled from 90 ± 1 to 144 ± 3 µm by changing pattern sizes (200, 300, and 400 µm). The shell thickness gradually increased from 12 ± 3 to 30 ± 6 µm by adjusting the endothelial cell seeding density. Finally, we fabricated the micro-tissue by fusion of the co-cultured spheroids, and the spheroids with the core-shell structure rapidly induced in vitro vessel-like network in 3 days. Thus, the position of endothelial cells in co-cultured spheroids may be an important factor for the modulation of the vascularization process, which can be useful for the production of 3D complex micro-tissues using spheroids as building blocks. STATEMENT OF SIGNIFICANCE: This manuscript describes our work on the fabrication of core-shell spheroids as building blocks to form 3D complex vascularized micro-tissue. Stem cells (human turbinate mesenchymal stem cells (hTMSCs) or adipose-derived stem cells (ADSCs)) and endothelial cells formed layered micro-sized cell sheets (µCSs) on micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids (core - stem cells, shell - endothelial cells), irrespective of stem cell type. In addition, the size and shell thickness of the core-shell spheroids were controlled by modifying pattern size and endothelial cell seeding density. We fabricated the vascularized micro-tissue by fusion of the spheroids and demonstrated that the spheroids with a core-shell structure rapidly induced vessel-like network.
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Affiliation(s)
- Eun Mi Kim
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea; BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University
| | - Yu Bin Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea; BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University
| | - Se-Jeong Kim
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea; BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University
| | - Jaesung Park
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea; BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University
| | - Jinkyu Lee
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea; BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University
| | - Sung Won Kim
- Department of Pathology, The Catholic University of Korea, College of Medicine, Seoul, Republic of Korea
| | - Hansoo Park
- School of Integrative Engineering, College of Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 06974, Republic of Korea.
| | - Heungsoo Shin
- Department of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea; BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University.
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Song L, Yuan X, Jones Z, Griffin K, Zhou Y, Ma T, Li Y. Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues. Sci Rep 2019; 9:5977. [PMID: 30979929 PMCID: PMC6461701 DOI: 10.1038/s41598-019-42439-9] [Citation(s) in RCA: 103] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Accepted: 04/01/2019] [Indexed: 02/07/2023] Open
Abstract
Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.
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Affiliation(s)
- Liqing Song
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA
| | - Xuegang Yuan
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA
| | - Zachary Jones
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA
| | - Kyle Griffin
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA
| | - Yi Zhou
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA
| | - Teng Ma
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA.
| | - Yan Li
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA.
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Studying Heterotypic Cell⁻Cell Interactions in the Human Brain Using Pluripotent Stem Cell Models for Neurodegeneration. Cells 2019; 8:cells8040299. [PMID: 30939814 PMCID: PMC6523455 DOI: 10.3390/cells8040299] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Revised: 03/26/2019] [Accepted: 03/29/2019] [Indexed: 02/08/2023] Open
Abstract
Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of the human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes (i.e., the tissue resident mesenchymal stromal cells), astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. In addition, most cortical organoids lack a microglia component, the resident immune cells in the brain. Impairment of the blood-brain barrier caused by improper crosstalk between neural cells and vascular cells is associated with many neurodegenerative disorders. Mesenchymal stem cells (MSCs), with a phenotype overlapping with pericytes, have promotion effects on neurogenesis and angiogenesis, which are mainly attributed to secreted growth factors and extracellular matrices. As the innate macrophages of the central nervous system, microglia regulate neuronal activities and promote neuronal differentiation by secreting neurotrophic factors and pro-/anti-inflammatory molecules. Neuronal-microglia interactions mediated by chemokines signaling can be modulated in vitro for recapitulating microglial activities during neurodegenerative disease progression. In this review, we discussed the cellular interactions and the physiological roles of neural cells with other cell types including endothelial cells and microglia based on iPSC models. The therapeutic roles of MSCs in treating neural degeneration and pathological roles of microglia in neurodegenerative disease progression were also discussed.
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Dechristé G, Fehrenbach J, Griseti E, Lobjois V, Poignard C. Viscoelastic modeling of the fusion of multicellular tumor spheroids in growth phase. J Theor Biol 2018; 454:102-109. [DOI: 10.1016/j.jtbi.2018.05.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Revised: 05/03/2018] [Accepted: 05/04/2018] [Indexed: 01/07/2023]
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Xu Y, Hu Y, Liu C, Yao H, Liu B, Mi S. A Novel Strategy for Creating Tissue-Engineered Biomimetic Blood Vessels Using 3D Bioprinting Technology. MATERIALS 2018; 11:ma11091581. [PMID: 30200455 PMCID: PMC6163305 DOI: 10.3390/ma11091581] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Revised: 08/22/2018] [Accepted: 08/27/2018] [Indexed: 02/07/2023]
Abstract
In this work, a novel strategy was developed to fabricate prevascularized cell-layer blood vessels in thick tissues and small-diameter blood vessel substitutes using three-dimensional (3D) bioprinting technology. These thick vascularized tissues were comprised of cells, a decellularized extracellular matrix (dECM), and a vasculature of multilevel sizes and multibranch architectures. Pluronic F127 (PF 127) was used as a sacrificial material for the formation of the vasculature through a multi-nozzle 3D bioprinting system. After printing, Pluronic F127 was removed to obtain multilevel hollow channels for the attachment of human umbilical vein endothelial cells (HUVECs). To reconstruct functional small-diameter blood vessel substitutes, a supporting scaffold (SE1700) with a double-layer circular structure was first bioprinted. Human aortic vascular smooth muscle cells (HA-VSMCs), HUVECs, and human dermal fibroblasts–neonatal (HDF-n) were separately used to form the media, intima, and adventitia through perfusion into the corresponding location of the supporting scaffold. In particular, the dECM was used as the matrix of the small-diameter blood vessel substitutes. After culture in vitro for 48 h, fluorescent images revealed that cells maintained their viability and that the samples maintained structural integrity. In addition, we analyzed the mechanical properties of the printed scaffold and found that its elastic modulus approximated that of the natural aorta. These findings demonstrate the feasibility of fabricating different kinds of vessels to imitate the structure and function of the human vascular system using 3D bioprinting technology.
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Affiliation(s)
- Yuanyuan Xu
- Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China.
- Biomanufacturing Engineering Laboratory, Advanced Manufacturing Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
| | - Yingying Hu
- Biomanufacturing Engineering Laboratory, Advanced Manufacturing Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
| | - Changyong Liu
- Additive Manufacturing Research Institute, College of Mechatronics and Control Engineering, Shenzhen University, Shenzhen 518060, China.
| | - Hongyi Yao
- Biomanufacturing Engineering Laboratory, Advanced Manufacturing Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
| | - Boxun Liu
- Department of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute, Shenzhen 518055, China.
| | - Shengli Mi
- Biomanufacturing Engineering Laboratory, Advanced Manufacturing Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
- Open FIESTA Center, Tsinghua University, Shenzhen 518055, China.
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Parfenov VA, Koudan EV, Bulanova EA, Karalkin PA, DAS Pereira F, Norkin NE, Knyazeva AD, Gryadunova AA, Petrov OF, Vasiliev MM, Myasnikov MI, Chernikov VP, Kasyanov VA, Marchenkov AY, Brakke K, Khesuani YD, Demirci U, Mironov VA. Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly. Biofabrication 2018; 10:034104. [PMID: 29848793 DOI: 10.1088/1758-5090/aac900] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering.
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Affiliation(s)
- Vladislav A Parfenov
- Laboratory for Biotechnological Research '3D Bioprinting Solutions', Moscow, Russia
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Kim TY, Kofron CM, King ME, Markes AR, Okundaye AO, Qu Z, Mende U, Choi BR. Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts. PLoS One 2018; 13:e0196714. [PMID: 29715271 PMCID: PMC5929561 DOI: 10.1371/journal.pone.0196714] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2017] [Accepted: 04/18/2018] [Indexed: 12/13/2022] Open
Abstract
Multicellular spheroids generated through cellular self-assembly provide cytoarchitectural complexities of native tissue including three-dimensionality, extensive cell-cell contacts, and appropriate cell-extracellular matrix interactions. They are increasingly suggested as building blocks for larger engineered tissues to achieve shapes, organization, heterogeneity, and other biomimetic complexities. Application of these tissue culture platforms is of particular importance in cardiac research as the myocardium is comprised of distinct but intermingled cell types. Here, we generated scaffold-free 3D cardiac microtissue spheroids comprised of cardiac myocytes (CMs) and/or cardiac fibroblasts (CFs) and used them as building blocks to form larger microtissues with different spatial distributions of CMs and CFs. Characterization of fusing homotypic and heterotypic spheroid pairs revealed an important influence of CFs on fusion kinetics, but most strikingly showed rapid fusion kinetics between heterotypic pairs consisting of one CF and one CM spheroid, indicating that CMs and CFs self-sort in vitro into the intermixed morphology found in the healthy myocardium. We then examined electrophysiological integration of fused homotypic and heterotypic microtissues by mapping action potential propagation. Heterocellular elongated microtissues which recapitulate the disproportionate CF spatial distribution seen in the infarcted myocardium showed that action potentials propagate through CF volumes albeit with significant delay. Complementary computational modeling revealed an important role of CF sodium currents and the spatial distribution of the CM-CF boundary in action potential conduction through CF volumes. Taken together, this study provides useful insights for the development of complex, heterocellular engineered 3D tissue constructs and their engraftment via tissue fusion and has implications for arrhythmogenesis in cardiac disease and repair.
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Affiliation(s)
- Tae Yun Kim
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
| | - Celinda M. Kofron
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
- Center for Biomedical Engineering, School of Engineering, Brown University, Providence, RI, United States of America
| | - Michelle E. King
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
| | - Alexander R. Markes
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
- Division of Biology and Medicine, Brown University, Providence, RI, United States of America
| | - Amenawon O. Okundaye
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
- Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, United States of America
| | - Zhilin Qu
- Division of Cardiology, Department of Medicine, University of California, Los Angeles, CA, United States of America
| | - Ulrike Mende
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
| | - Bum-Rak Choi
- Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI, United States of America
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Abstract
Biofabrication of tissue analogues is aspiring to become a disruptive technology capable to solve standing biomedical problems, from generation of improved tissue models for drug testing to alleviation of the shortage of organs for transplantation. Arguably, the most powerful tool of this revolution is bioprinting, understood as the assembling of cells with biomaterials in three‐dimensional structures. It is less appreciated, however, that bioprinting is not a uniform methodology, but comprises a variety of approaches. These can be broadly classified in two categories, based on the use or not of supporting biomaterials (known as “scaffolds,” usually printable hydrogels also called “bioinks”). Importantly, several limitations of scaffold‐dependent bioprinting can be avoided by the “scaffold‐free” methods. In this overview, we comparatively present these approaches and highlight the rapidly evolving scaffold‐free bioprinting, as applied to cardiovascular tissue engineering.
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Affiliation(s)
- Nicanor I Moldovan
- Departments of Biomedical Engineering and Ophthalmology, 3D Bioprinting Core, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA
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Thomas D, O'Brien T, Pandit A. Toward Customized Extracellular Niche Engineering: Progress in Cell-Entrapment Technologies. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2018; 30:1703948. [PMID: 29194781 DOI: 10.1002/adma.201703948] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/15/2017] [Revised: 09/12/2017] [Indexed: 06/07/2023]
Abstract
The primary aim in tissue engineering is to repair, replace, and regenerate dysfunctional tissues to restore homeostasis. Cell delivery for repair and regeneration is gaining impetus with our understanding of constructing tissue-like environments. However, the perpetual challenge is to identify innovative materials or re-engineer natural materials to model cell-specific tissue-like 3D modules, which can seamlessly integrate and restore functions of the target organ. To devise an optimal functional microenvironment, it is essential to define how simple is complex enough to trigger tissue regeneration or restore cellular function. Here, the purposeful transition of cell immobilization from a cytoprotection point of view to that of a cell-instructive approach is examined, with advances in the understanding of cell-material interactions in a 3D context, and with a view to further application of the knowledge for the development of newer and complex hierarchical tissue assemblies for better examination of cell behavior and offering customized cell-based therapies for tissue engineering.
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Affiliation(s)
- Dilip Thomas
- Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Galway, Ireland
- Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland
- Cardiovascular Institute, Stanford University, Palo Alto, CA, 94305, USA
| | - Timothy O'Brien
- Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland
| | - Abhay Pandit
- Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Galway, Ireland
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