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Hammad M, Dugué J, Maubert E, Baugé C, Boumédiene K. Decellularized apple hypanthium as a plant-based biomaterial for cartilage regeneration in vitro: a comparative study of progenitor cell types and environmental conditions. J Biol Eng 2025; 19:38. [PMID: 40264116 PMCID: PMC12012941 DOI: 10.1186/s13036-025-00502-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2024] [Accepted: 04/04/2025] [Indexed: 04/24/2025] Open
Abstract
BACKGROUND Decellularized plant tissues have been shown to enhance the integration and proliferation of human cells, demonstrating biocompatibility. These tissues are now being considered as valuable biomaterials for tissue engineering due to their diverse architectures and favorable cytocompatibility. In this study, we assessed decellularized apple hypanthium as a potential biomaterial for generating cartilage-like structures, utilizing four different progenitor cell types and varying environmental conditions in vitro. RESULTS Cell viability assays indicated integration and cell proliferation. Histological staining and gene expression analyses confirmed the synthesis and deposition of a cartilaginous extracellular matrix. Notably, hypoxia had varying effects on chondrogenesis based on the cell type. Among the progenitor cells evaluated, those derived from auricular perichondrium were particularly promising, as they differentiated into chondrocytes without requiring a low-oxygen environment. Additionally, our findings demonstrated that apple-derived biomaterials outperformed microencapsulation in alginate beads in promoting chondrogenesis. CONCLUSION These results highlight the potential of plant-based biomaterials for the development of implantable devices for cartilage regeneration and suggest broader applications in tissue engineering and future clinical endeavors.
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Affiliation(s)
- Mira Hammad
- Laboratoire BioConnect UR 7451, Université de Caen Normandie, Esplanade de la paix CS14032, Caen, 14032 Caen Cedex 5, France
- Fédération Hospitalo Universitaire SURFACE, Amiens, Caen, Rouen, France
| | - Justin Dugué
- Laboratoire BioConnect UR 7451, Université de Caen Normandie, Esplanade de la paix CS14032, Caen, 14032 Caen Cedex 5, France
- Fédération Hospitalo Universitaire SURFACE, Amiens, Caen, Rouen, France
- Service ORL et chirurgie Cervico-faciale, CHU de Caen, Caen, France
| | - Eric Maubert
- Phind Inserm UMR-S 1237, Université de Caen Normandie, Caen, France
| | - Catherine Baugé
- Laboratoire BioConnect UR 7451, Université de Caen Normandie, Esplanade de la paix CS14032, Caen, 14032 Caen Cedex 5, France
- Fédération Hospitalo Universitaire SURFACE, Amiens, Caen, Rouen, France
| | - Karim Boumédiene
- Laboratoire BioConnect UR 7451, Université de Caen Normandie, Esplanade de la paix CS14032, Caen, 14032 Caen Cedex 5, France.
- Fédération Hospitalo Universitaire SURFACE, Amiens, Caen, Rouen, France.
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García-Gareta E, Calderón-Villalba A, Alamán-Díez P, Costa CG, Guerrero PE, Mur C, Flores AR, Jurjo NO, Sancho P, Pérez MÁ, García-Aznar JM. Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma. Eur J Cell Biol 2024; 103:151396. [PMID: 38359522 DOI: 10.1016/j.ejcb.2024.151396] [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: 08/30/2023] [Revised: 01/25/2024] [Accepted: 02/10/2024] [Indexed: 02/17/2024] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.
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Affiliation(s)
- Elena García-Gareta
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain; Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza, Aragon, Spain; Division of Biomaterials & Tissue Engineering, UCL Eastman Dental Institute, University College London, London, United Kingdom.
| | - Alejandro Calderón-Villalba
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Pilar Alamán-Díez
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Carlos Gracia Costa
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Pedro Enrique Guerrero
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Carlota Mur
- Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Ana Rueda Flores
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Nerea Olivera Jurjo
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain
| | - Patricia Sancho
- Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza, Aragon, Spain
| | - María Ángeles Pérez
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain; Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza, Aragon, Spain
| | - José Manuel García-Aznar
- Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon, Spain; Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza, Aragon, Spain
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Golebiowska AA, Intravaia JT, Sathe VM, Kumbar SG, Nukavarapu SP. Decellularized extracellular matrix biomaterials for regenerative therapies: Advances, challenges and clinical prospects. Bioact Mater 2024; 32:98-123. [PMID: 37927899 PMCID: PMC10622743 DOI: 10.1016/j.bioactmat.2023.09.017] [Citation(s) in RCA: 36] [Impact Index Per Article: 36.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 09/21/2023] [Accepted: 09/25/2023] [Indexed: 11/07/2023] Open
Abstract
Tissue engineering and regenerative medicine have shown potential in the repair and regeneration of tissues and organs via the use of engineered biomaterials and scaffolds. However, current constructs face limitations in replicating the intricate native microenvironment and achieving optimal regenerative capacity and functional recovery. To address these challenges, the utilization of decellularized tissues and cell-derived extracellular matrix (ECM) has emerged as a promising approach. These biocompatible and bioactive biomaterials can be engineered into porous scaffolds and grafts that mimic the structural and compositional aspects of the native tissue or organ microenvironment, both in vitro and in vivo. Bioactive dECM materials provide a unique tissue-specific microenvironment that can regulate and guide cellular processes, thereby enhancing regenerative therapies. In this review, we explore the emerging frontiers of decellularized tissue-derived and cell-derived biomaterials and bio-inks in the field of tissue engineering and regenerative medicine. We discuss the need for further improvements in decellularization methods and techniques to retain structural, biological, and physicochemical characteristics of the dECM products in a way to mimic native tissues and organs. This article underscores the potential of dECM biomaterials to stimulate in situ tissue repair through chemotactic effects for the development of growth factor and cell-free tissue engineering strategies. The article also identifies the challenges and opportunities in developing sterilization and preservation methods applicable for decellularized biomaterials and grafts and their translation into clinical products.
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Affiliation(s)
| | - Jonathon T. Intravaia
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA
| | - Vinayak M. Sathe
- Department of Orthopaedic Surgery, University of Connecticut Health, Farmington, CT, 06032, USA
| | - Sangamesh G. Kumbar
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA
- Department of Materials Science & Engineering, University of Connecticut, Storrs, CT, 06269, USA
- Department of Orthopaedic Surgery, University of Connecticut Health, Farmington, CT, 06032, USA
| | - Syam P. Nukavarapu
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA
- Department of Materials Science & Engineering, University of Connecticut, Storrs, CT, 06269, USA
- Department of Orthopaedic Surgery, University of Connecticut Health, Farmington, CT, 06032, USA
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Dhandapani V, Vermette P. Decellularized bladder as scaffold to support proliferation and functionality of insulin-secreting pancreatic cells. J Biomed Mater Res B Appl Biomater 2023; 111:1890-1902. [PMID: 37306142 DOI: 10.1002/jbm.b.35292] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Revised: 05/07/2023] [Accepted: 05/31/2023] [Indexed: 06/13/2023]
Abstract
Loss in the number or function of insulin-producing β-cells in pancreatic islets has been associated with diabetes mellitus. Although islet transplantation can be an alternative treatment, complications such as apoptosis, ischaemia and loss of viability have been reported. The use of decellularized organs as scaffolds in tissue engineering is of interest owing to the unique ultrastructure and composition of the extracellular matrix (ECM) believed to act on tissue regeneration. In this study, a cell culture system has been designed to study the effect of decellularized porcine bladder pieces on INS-1 cells, a cell line secreting insulin in response to glucose stimulation. Porcine bladders were decellularized using two techniques: a detergent-containing and a detergent-free methods. The resulting ECMs were characterized for the removal of both cells and dsDNA. INS-1 cells were not viable on ECM produced using detergent (i.e., sodium dodecyl sulfate). INS-1 cells were visualized following 7 days of culture on detergent-free decellularized bladders using a cell viability and metabolism assay (MTT) and cell proliferation quantified (CyQUANT™ NF Cell Proliferation Assay). Further, glucose-stimulated insulin secretion and immunostaining confirmed that cells were functional in response to glucose stimulation, as well as they expressed insulin and interacted with the detergent-free produced ECM, respectively.
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Affiliation(s)
- Vignesh Dhandapani
- Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, Canada
- Centre de recherche du CHUS, Faculté de médecine et des sciences de la santé, Sherbrooke, Canada
| | - Patrick Vermette
- Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, Canada
- Centre de recherche du CHUS, Faculté de médecine et des sciences de la santé, Sherbrooke, Canada
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Allu I, Sahi AK, Koppadi M, Gundu S, Sionkowska A. Decellularization Techniques for Tissue Engineering: Towards Replicating Native Extracellular Matrix Architecture in Liver Regeneration. J Funct Biomater 2023; 14:518. [PMID: 37888183 PMCID: PMC10607724 DOI: 10.3390/jfb14100518] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Revised: 10/09/2023] [Accepted: 10/10/2023] [Indexed: 10/28/2023] Open
Abstract
The process of tissue regeneration requires the utilization of a scaffold, which serves as a structural framework facilitating cellular adhesion, proliferation, and migration within a physical environment. The primary aim of scaffolds in tissue engineering is to mimic the structural and functional properties of the extracellular matrix (ECM) in the target tissue. The construction of scaffolds that accurately mimic the architecture of the extracellular matrix (ECM) is a challenging task, primarily due to the intricate structural nature and complex composition of the ECM. The technique of decellularization has gained significant attention in the field of tissue regeneration because of its ability to produce natural scaffolds by removing cellular and genetic components from the extracellular matrix (ECM) while preserving its structural integrity. The present study aims to investigate the various decellularization techniques employed for the purpose of isolating the extracellular matrix (ECM) from its native tissue. Additionally, a comprehensive comparison of these methods will be presented, highlighting their respective advantages and disadvantages. The primary objective of this study is to gain a comprehensive understanding of the anatomical and functional features of the native liver, as well as the prevalence and impact of liver diseases. Additionally, this study aims to identify the limitations and difficulties associated with existing therapeutic methods for liver diseases. Furthermore, the study explores the potential of tissue engineering techniques in addressing these challenges and enhancing liver performance. By investigating these aspects, this research field aims to contribute to the advancement of liver disease treatment and management.
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Affiliation(s)
- Ishita Allu
- Department of Biomedical Engineering, University College of Engineering (UCE), Osmania University, Hyderabad 500007, India; (I.A.); (M.K.)
| | - Ajay Kumar Sahi
- School of Medicine, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA;
| | - Meghana Koppadi
- Department of Biomedical Engineering, University College of Engineering (UCE), Osmania University, Hyderabad 500007, India; (I.A.); (M.K.)
| | - Shravanya Gundu
- Department of Biomedical Engineering, University College of Engineering (UCE), Osmania University, Hyderabad 500007, India; (I.A.); (M.K.)
| | - Alina Sionkowska
- Faculty of Chemistry, Nicolaus Copernicus University in Torun, Jurija Gagarina 11, 87-100 Torun, Poland
- Faculty of Health Sciences, Calisia University, Nowy Świat 4, 62-800 Kalisz, Poland
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Mir TA, Alzhrani A, Nakamura M, Iwanaga S, Wani SI, Altuhami A, Kazmi S, Arai K, Shamma T, Obeid DA, Assiri AM, Broering DC. Whole Liver Derived Acellular Extracellular Matrix for Bioengineering of Liver Constructs: An Updated Review. Bioengineering (Basel) 2023; 10:1126. [PMID: 37892856 PMCID: PMC10604736 DOI: 10.3390/bioengineering10101126] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2023] [Revised: 09/13/2023] [Accepted: 09/15/2023] [Indexed: 10/29/2023] Open
Abstract
Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.
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Affiliation(s)
- Tanveer Ahmed Mir
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
| | - Alaa Alzhrani
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
- Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21423, Saudi Arabia
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia
| | - Makoto Nakamura
- Division of Biomedical System Engineering, Graduate School of Science and Engineering for Education, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan; (M.N.); (S.I.)
| | - Shintaroh Iwanaga
- Division of Biomedical System Engineering, Graduate School of Science and Engineering for Education, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan; (M.N.); (S.I.)
| | - Shadil Ibrahim Wani
- Division of Biomedical System Engineering, Graduate School of Science and Engineering for Education, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan; (M.N.); (S.I.)
| | - Abdullah Altuhami
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
| | - Shadab Kazmi
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
- Department of Child Health, School of Medicine, University of Missouri, Columbia, MO 65212, USA
| | - Kenchi Arai
- Department of Clinical Biomaterial Applied Science, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan
| | - Talal Shamma
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
| | - Dalia A. Obeid
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
| | - Abdullah M. Assiri
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia
| | - Dieter C. Broering
- Laboratory of Tissue/Organ Bioengineering & BioMEMS, Organ Transplant Centre of Excellence, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia (T.S.)
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia
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Data K, Kulus M, Ziemak H, Chwarzyński M, Piotrowska-Kempisty H, Bukowska D, Antosik P, Mozdziak P, Kempisty B. Decellularization of Dense Regular Connective Tissue-Cellular and Molecular Modification with Applications in Regenerative Medicine. Cells 2023; 12:2293. [PMID: 37759515 PMCID: PMC10528602 DOI: 10.3390/cells12182293] [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: 07/13/2023] [Revised: 08/31/2023] [Accepted: 09/14/2023] [Indexed: 09/29/2023] Open
Abstract
Healing of dense regular connective tissue, due to a high fiber-to-cell ratio and low metabolic activity and regeneration potential, frequently requires surgical implantation or reconstruction with high risk of reinjury. An alternative to synthetic implants is using bioscaffolds obtained through decellularization, a process where the aim is to extract cells from the tissue while preserving the tissue-specific native molecular structure of the ECM. Proteins, lipids, nucleic acids and other various extracellular molecules are largely involved in differentiation, proliferation, vascularization and collagen fibers deposit, making them the crucial processes in tissue regeneration. Because of the multiple possible forms of cell extraction, there is no standardized protocol in dense regular connective tissue (DRCT). Many modifications of the structure, shape and composition of the bioscaffold have also been described to improve the therapeutic result following the implantation of decellularized connective tissue. The available data provide a valuable source of crucial information. However, the wide spectrum of decellularization makes it important to understand the key aspects of bioscaffolds relative to their potential use in tissue regeneration.
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Affiliation(s)
- Krzysztof Data
- Division of Anatomy, Department of Human Morphology and Embryology, Wroclaw Medical University, 50-368 Wroclaw, Poland
| | - Magdalena Kulus
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
| | - Hanna Ziemak
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
| | - Mikołaj Chwarzyński
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
| | - Hanna Piotrowska-Kempisty
- Department of Toxicology, Poznan University of Medical Sciences, 60-631 Poznan, Poland
- Department of Basic and Preclinical Sciences, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
| | - Dorota Bukowska
- Department of Diagnostics and Clinical Sciences, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
| | - Paweł Antosik
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
| | - Paul Mozdziak
- Physiolgy Graduate Faculty, North Carolina State University, Raleigh, NC 27695, USA
- Prestage Department of Poultry Sciences, North Carolina State University, Raleigh, NC 27695, USA
| | - Bartosz Kempisty
- Division of Anatomy, Department of Human Morphology and Embryology, Wroclaw Medical University, 50-368 Wroclaw, Poland
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 87-100 Torun, Poland
- Physiolgy Graduate Faculty, North Carolina State University, Raleigh, NC 27695, USA
- Department of Obstetrics and Gynecology, University Hospital and Masaryk University, 601 77 Brno, Czech Republic
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Bengur FB, Chen L, Schilling BK, Komatsu C, Figlioli GM, Marra KG, Kokai LE, Solari MG. Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate-Based Protocols. J Reconstr Microsurg 2023; 39:493-501. [PMID: 36584695 DOI: 10.1055/s-0042-1760110] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND Free tissue transfer to cover complex wounds with exposed critical structures results in donor-site morbidity. Perfusion decellularization and recellularization of vascularized composite tissues is an active area of research to fabricate complex constructs without a donor site. Sodium dodecyl sulfate (SDS)-based protocols remain the predominant choice for decellularization despite the deleterious effects on tissue ultrastructure and capillary networks. We aimed to develop an automated decellularization process and compare different SDS perfusion times to optimize the protocol. METHODS A three-dimensional-printed closed-system bioreactor capable of continuously perfusing fluid through the vasculature was used for decellularization. The artery and vein of rat epigastric fasciocutaneous free flaps were cannulated and connected to the bioreactor. Protocols had varying durations of 1% SDS solution (3, 5, and 10 days) followed by 1 day of 1% Triton X-100 and 1 day of 1x phosphate-buffered saline. The residual DNA was quantified. Microarchitecture of the constructs was assessed with histology, and the vascular network was visualized for qualitative assessment. RESULTS The structural integrity and the microarchitecture of the extracellular matrix was preserved in the 3- and 5-day SDS perfusion groups; however, the subcutaneous tissue of the 10-day protocol lost its structure. Collagen and elastin structures of the pedicle vessels were not compromised by the decellularization process. Five-day SDS exposure group had the least residual DNA content (p < 0.001). Across all protocols, skin consistently had twice as much residual DNA over the subcutaneous tissues. CONCLUSION A compact and integrated bioreactor can automate decellularization of free flaps to bioengineer regenerative constructs for future use in reconstruction of complex defects. A decellularization protocol with 5 days of 1% SDS exposure was the most successful to keep the residual DNA content at a minimum while preserving the structural integrity of the tissues.
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Affiliation(s)
- Fuat Baris Bengur
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Lei Chen
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Benjamin K Schilling
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Chiaki Komatsu
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Grace M Figlioli
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Kacey G Marra
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Lauren E Kokai
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Mario G Solari
- Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
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9
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Cardinale V, Lanthier N, Baptista PM, Carpino G, Carnevale G, Orlando G, Angelico R, Manzia TM, Schuppan D, Pinzani M, Alvaro D, Ciccocioppo R, Uygun BE. Cell transplantation-based regenerative medicine in liver diseases. Stem Cell Reports 2023; 18:1555-1572. [PMID: 37557073 PMCID: PMC10444572 DOI: 10.1016/j.stemcr.2023.06.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2022] [Revised: 06/11/2023] [Accepted: 06/12/2023] [Indexed: 08/11/2023] Open
Abstract
This review aims to evaluate the current preclinical state of liver bioengineering, the clinical context for liver cell therapies, the cell sources, the delivery routes, and the results of clinical trials for end-stage liver disease. Different clinical settings, such as inborn errors of metabolism, acute liver failure, chronic liver disease, liver cirrhosis, and acute-on-chronic liver failure, as well as multiple cellular sources were analyzed; namely, hepatocytes, hepatic progenitor cells, biliary tree stem/progenitor cells, mesenchymal stromal cells, and macrophages. The highly heterogeneous clinical scenario of liver disease and the availability of multiple cellular sources endowed with different biological properties make this a multidisciplinary translational research challenge. Data on each individual liver disease and more accurate endpoints are urgently needed, together with a characterization of the regenerative pathways leading to potential therapeutic benefit. Here, we critically review these topics and identify related research needs and perspectives in preclinical and clinical settings.
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Affiliation(s)
- Vincenzo Cardinale
- Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy.
| | - Nicolas Lanthier
- Service d'Hépato-gastroentérologie, Cliniques Universitaires Saint-Luc, Laboratory of Hepatogastroenterology, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium
| | - Pedro M Baptista
- Instituto de Investigación Sanitaria Aragón (IIS Aragón), Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas (CIBERehd), Madrid, Spain; Fundación ARAID, Zaragoza, Spain; Department of Biomedical and Aerospace Engineering, Universidad Carlos III de Madrid, Madrid, Spain
| | - Guido Carpino
- Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, Sapienza University of Rome, Italy
| | - Gianluca Carnevale
- Department of Surgery, Medicine, Dentistry, and Morphological Sciences with Interest in Transplant, Oncology, and Regenerative Medicine, University of Modena and Reggio Emilia, 41125 Modena, Italy
| | - Giuseppe Orlando
- Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, NC, USA
| | - Roberta Angelico
- Hepatobiliary Surgery and Transplant Unit, Department of Surgical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Tommaso Maria Manzia
- Hepatobiliary Surgery and Transplant Unit, Department of Surgical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Detlef Schuppan
- Institute of Translational Immunology, Research Center for Immune Therapy, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany; Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Massimo Pinzani
- UCL Institute for Liver and Digestive Health, Division of Medicine, Royal Free Hospital, London, UK
| | - Domenico Alvaro
- Department of Translation and Precision Medicine, "Sapienza" University of Rome, Rome, Italy
| | - Rachele Ciccocioppo
- Gastroenterology Unit, Department of Medicine, A.O.U.I. Policlinico G.B. Rossi & University of Verona, Verona, Italy.
| | - Basak E Uygun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, Boston, MA 02114, USA; Department of Surgery, Massachusetts General Hospital, Boston, MA 02114, USA.
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10
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Zhe M, Wu X, Yu P, Xu J, Liu M, Yang G, Xiang Z, Xing F, Ritz U. Recent Advances in Decellularized Extracellular Matrix-Based Bioinks for 3D Bioprinting in Tissue Engineering. MATERIALS (BASEL, SWITZERLAND) 2023; 16:3197. [PMID: 37110034 PMCID: PMC10143913 DOI: 10.3390/ma16083197] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 03/30/2023] [Accepted: 04/15/2023] [Indexed: 06/19/2023]
Abstract
In recent years, three-dimensional (3D) bioprinting has been widely utilized as a novel manufacturing technique by more and more researchers to construct various tissue substitutes with complex architectures and geometries. Different biomaterials, including natural and synthetic materials, have been manufactured into bioinks for tissue regeneration using 3D bioprinting. Among the natural biomaterials derived from various natural tissues or organs, the decellularized extracellular matrix (dECM) has a complex internal structure and a variety of bioactive factors that provide mechanistic, biophysical, and biochemical signals for tissue regeneration and remodeling. In recent years, more and more researchers have been developing the dECM as a novel bioink for the construction of tissue substitutes. Compared with other bioinks, the various ECM components in dECM-based bioink can regulate cellular functions, modulate the tissue regeneration process, and adjust tissue remodeling. Therefore, we conducted this review to discuss the current status of and perspectives on dECM-based bioinks for bioprinting in tissue engineering. In addition, the various bioprinting techniques and decellularization methods were also discussed in this study.
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Affiliation(s)
- Man Zhe
- Animal Experiment Center, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Xinyu Wu
- West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
| | - Peiyun Yu
- LIMES Institute, Department of Molecular Brain Physiology and Behavior, University of Bonn, Carl-Troll-Str. 31, 53115 Bonn, Germany
| | - Jiawei Xu
- Orthopedic Research Institute, Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Ming Liu
- Orthopedic Research Institute, Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Guang Yang
- Animal Experiment Center, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Zhou Xiang
- Orthopedic Research Institute, Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Fei Xing
- Department of Orthopaedics and Traumatology, Biomatics Group, University Medical Center of the Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz, Germany
| | - Ulrike Ritz
- Department of Orthopaedics and Traumatology, Biomatics Group, University Medical Center of the Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz, Germany
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11
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Bonciog DD, Lascu MR, Mâțiu-Iovan L, Ordodi VL. Automation and Optimization of Rat Heart Decellularization Using a Vibrating Fluid Column. SENSORS (BASEL, SWITZERLAND) 2023; 23:4045. [PMID: 37112386 PMCID: PMC10140852 DOI: 10.3390/s23084045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/14/2023] [Revised: 04/10/2023] [Accepted: 04/15/2023] [Indexed: 06/19/2023]
Abstract
This paper presents the validation of a software application to optimize the discoloration process in simulated hearts and to automate and determine the final moment of decellularization in rat hearts using a vibrating fluid column. The implemented algorithm specifically for the automated verification of a simulated heart's discoloration process was optimized in this study. Initially, we used a latex balloon containing enough dye to reach the opacity of a heart. The complete discoloration process corresponds to complete decellularization. The developed software automatically detects the complete discoloration of a simulated heart. Finally, the process stops automatically. Another goal was to optimize the Langendorff-type experimental apparatus, which is pressure-controlled and equipped with a vibrating fluid column that shortens the decellularization time by mechanically acting directly on cell membranes. Control experiments were performed with the designed experimental device and the vibrating liquid column using different decellularization protocols for hearts taken from rats. In this work, we used a commonly utilized solution based on sodium dodecyl sulfate. Ultraviolet spectrophotometry was used to measure the evolution of the dye concentration in the simulated hearts and, similarly, to determine the concentrations of deoxyribonucleic acid (DNA) and proteins in the rat hearts.
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Affiliation(s)
- Dumitru-Daniel Bonciog
- Measurements and Optical Electronics Department, Politehnica University Timisoara, 300006 Timisoara, Romania; (D.-D.B.); (M.-R.L.); (L.M.-I.)
| | - Mihaela-Ruxandra Lascu
- Measurements and Optical Electronics Department, Politehnica University Timisoara, 300006 Timisoara, Romania; (D.-D.B.); (M.-R.L.); (L.M.-I.)
| | - Liliana Mâțiu-Iovan
- Measurements and Optical Electronics Department, Politehnica University Timisoara, 300006 Timisoara, Romania; (D.-D.B.); (M.-R.L.); (L.M.-I.)
| | - Valentin Laurențiu Ordodi
- Chemistry and Engineering of Organic and Natural Compounds Department, University Politehnica Timisoara, 300006 Timisoara, Romania
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12
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Zhu L, Yuhan J, Yu H, Zhang B, Huang K, Zhu L. Decellularized Extracellular Matrix for Remodeling Bioengineering Organoid's Microenvironment. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2023:e2207752. [PMID: 36929582 DOI: 10.1002/smll.202207752] [Citation(s) in RCA: 45] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 02/17/2023] [Indexed: 06/18/2023]
Abstract
Over the past decade, stem cell- and tumor-derived organoids are the most promising models in developmental biology and disease modeling, respectively. The matrix is one of three main elements in the construction of an organoid and the most important module of its extracellular microenvironment. However, the source of the currently available commercial matrix, Matrigel, limits the application of organoids in clinical medicine. It is worth investigating whether the original decellularized extracellular matrix (dECM) can be exploited as the matrix of organoids and improving organoid construction are very important. In this review, tissue decellularization protocols and the characteristics of decellularization methods, the mechanical support and biological cues of extraccellular matrix (ECM), methods for construction of multifunctional dECM and responsive dECM hydrogel, and the potential applications of functional dECM are summarized. In addition, some expectations are provided for dECM as the matrix of organoids in clinical applications.
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Affiliation(s)
- Liye Zhu
- Key Laboratory of Precision Nutrition and Food Quality, Department of Nutrition and Health, China Agricultural University, Beijing, 100083, P. R. China
- College of Veterinary Medicine, China Agricultural University, Beijing, 100094, P. R. China
| | - Jieyu Yuhan
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, P. R. China
| | - Hao Yu
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, P. R. China
| | - Boyang Zhang
- Key Laboratory of Precision Nutrition and Food Quality, Department of Nutrition and Health, China Agricultural University, Beijing, 100083, P. R. China
| | - Kunlun Huang
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, P. R. China
| | - Longjiao Zhu
- Key Laboratory of Precision Nutrition and Food Quality, Department of Nutrition and Health, China Agricultural University, Beijing, 100083, P. R. China
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13
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Afzal Z, Huguet EL. Bioengineering liver tissue by repopulation of decellularised scaffolds. World J Hepatol 2023; 15:151-179. [PMID: 36926238 PMCID: PMC10011915 DOI: 10.4254/wjh.v15.i2.151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 11/22/2022] [Accepted: 02/15/2023] [Indexed: 02/24/2023] Open
Abstract
Liver transplantation is the only curative therapy for end stage liver disease, but is limited by the organ shortage, and is associated with the adverse consequences of immunosuppression. Repopulation of decellularised whole organ scaffolds with appropriate cells of recipient origin offers a theoretically attractive solution, allowing reliable and timely organ sourcing without the need for immunosuppression. Decellularisation methodologies vary widely but seek to address the conflicting objectives of removing the cellular component of tissues whilst keeping the 3D structure of the extra-cellular matrix intact, as well as retaining the instructive cell fate determining biochemicals contained therein. Liver scaffold recellularisation has progressed from small rodent in vitro studies to large animal in vivo perfusion models, using a wide range of cell types including primary cells, cell lines, foetal stem cells, and induced pluripotent stem cells. Within these models, a limited but measurable degree of physiologically significant hepatocyte function has been reported with demonstrable ammonia metabolism in vivo. Biliary repopulation and function have been restricted by challenges relating to the culture and propagations of cholangiocytes, though advances in organoid culture may help address this. Hepatic vasculature repopulation has enabled sustainable blood perfusion in vivo, but with cell types that would limit clinical applications, and which have not been shown to have the specific functions of liver sinusoidal endothelial cells. Minority cell groups such as Kupffer cells and stellate cells have not been repopulated. Bioengineering by repopulation of decellularised scaffolds has significantly progressed, but there remain significant experimental challenges to be addressed before therapeutic applications may be envisaged.
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Affiliation(s)
- Zeeshan Afzal
- Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Centre; Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Emmanuel Laurent Huguet
- Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Centre; Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
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14
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Borges MF, Maurmann N, Pranke P. Easy-to-Assembly System for Decellularization and Recellularization of Liver Grafts in a Bioreactor. MICROMACHINES 2023; 14:449. [PMID: 36838149 PMCID: PMC9962055 DOI: 10.3390/mi14020449] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 01/31/2023] [Accepted: 02/10/2023] [Indexed: 06/18/2023]
Abstract
Decellularization of organs creates an acellular scaffold, ideal for being repopulated by cells. In this work, a low-cost perfusion system was created to be used in the process of liver decellularization and as a bioreactor after recellularization. It consists of a glass chamber to house the organ coupled to a peristaltic pump to promote liquid flow through the organ vascular tree. The rats' liver decellularization was made with a solution of sodium dodecyl sulfate. The recellularization was made with 108 mesenchymal stromal/stem cells and cultivated for seven days. The decellularized matrices showed an absence of DNA while preserving the collagen and glycosaminoglycans quantities, confirming the efficiency of the process. The functional analyses showed a rise in lactate dehydrogenase levels occurring in the first days of the cultivation, suggesting that there is cell death in this period, which stabilized on the seventh day. Histological analysis showed conservation of the collagen web and some groups of cells next to the vessels. It was possible to establish a system for decellularization and a bioreactor to use for the recellularization method. It is easy to assemble, can be ready to use in little time and be easily sterilized.
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Affiliation(s)
- Maurício Felisberto Borges
- Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 90610-000, Brazil
| | - Natasha Maurmann
- Postgraduate Program in Physiology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 90050-170, Brazil
| | - Patricia Pranke
- Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 90610-000, Brazil
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15
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Toprakhisar B, Verfaillie CM, Kumar M. Advances in Recellularization of Decellularized Liver Grafts with Different Liver (Stem) Cells: Towards Clinical Applications. Cells 2023; 12:301. [PMID: 36672236 PMCID: PMC9856398 DOI: 10.3390/cells12020301] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Revised: 12/22/2022] [Accepted: 12/28/2022] [Indexed: 01/15/2023] Open
Abstract
Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized.
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Affiliation(s)
- Burak Toprakhisar
- Stem Cell Institute, Department of Stem Cell and Developmental Biology, KU Leuven, 3000 Leuven, Belgium
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16
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Jambar Nooshin B, Tayebi T, Babajani A, Khani MM, Niknejad H. Effects of Different Perfusing Routes through The Portal Vein, Hepatic Vein, and Biliary Duct on Whole Rat Liver Decellularization. CELL JOURNAL 2023; 25:35-44. [PMID: 36680482 PMCID: PMC9868438 DOI: 10.22074/cellj.2022.557600.1081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Revised: 09/11/2022] [Accepted: 10/04/2022] [Indexed: 01/22/2023]
Abstract
OBJECTIVE Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes. MATERIALS AND METHODS In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines. RESULTS Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H and E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold. CONCLUSION The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.
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Affiliation(s)
- Bahram Jambar Nooshin
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Tahereh Tayebi
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Amirhesam Babajani
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Mehdi Khani
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
| | - Hassan Niknejad
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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17
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Campinoti S, Almeida B, Goudarzi N, Bencina S, Grundland Freile F, McQuitty C, Natarajan D, Cox IJ, Le Guennec A, Khati V, Gaudenzi G, Gramignoli R, Urbani L. Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells. J Tissue Eng 2023; 14:20417314231219813. [PMID: 38143931 PMCID: PMC10748678 DOI: 10.1177/20417314231219813] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Accepted: 11/25/2023] [Indexed: 12/26/2023] Open
Abstract
Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells' maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.
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Affiliation(s)
- Sara Campinoti
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
| | - Bruna Almeida
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
| | - Negin Goudarzi
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
| | - Stefan Bencina
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Solna, Sweden
| | - Fabio Grundland Freile
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Department of Medical and Molecular Genetics, School of Basic and Medical Bioscience, Faculty of Life Science and Medicine, King’s College London, London, UK
| | - Claire McQuitty
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
| | - Dipa Natarajan
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
| | - I Jane Cox
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
| | - Adrien Le Guennec
- Centre for Biomolecular Spectroscopy, Randall Centre for Cell and Molecular Biophysics, Kings College London, London, UK
| | - Vamakshi Khati
- Science for Life Laboratory, Division of Nanobiotechnology, Department of Protein Science, KTH Royal Institute of Technology, Solna, Sweden
| | - Giulia Gaudenzi
- Department of Global Public Health, Karolinska Institutet, Solna, Sweden
| | - Roberto Gramignoli
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Solna, Sweden
- Department of Pathology and Cancer Diagnostics, Karolinska University Hospital, Huddinge, Sweden
| | - Luca Urbani
- The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK
- Faculty of Life Sciences and Medicine, King’s College London, London, UK
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18
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Singh G, Senapati S, Satpathi S, Behera PK, Das B, Nayak B. Establishment of decellularized extracellular matrix scaffold derived from caprine pancreas as a novel alternative template over porcine pancreatic scaffold for prospective biomedical application. FASEB J 2022; 36:e22574. [PMID: 36165227 DOI: 10.1096/fj.202200807r] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2022] [Revised: 08/25/2022] [Accepted: 09/16/2022] [Indexed: 11/11/2022]
Abstract
In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.
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Affiliation(s)
- Garima Singh
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, India
| | - Shantibhusan Senapati
- Tumor Microenvironment and Animal Models Laboratory, Institute of Life Sciences, Bhubaneswar, India
| | | | | | - Biswajit Das
- Tumor Microenvironment and Animal Models Laboratory, Institute of Life Sciences, Bhubaneswar, India
| | - Bismita Nayak
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, India
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19
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McDuffie D, Barr D, Agarwal A, Thomas E. Physiologically relevant microsystems to study viral infection in the human liver. Front Microbiol 2022; 13:999366. [PMID: 36246284 PMCID: PMC9555087 DOI: 10.3389/fmicb.2022.999366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 08/29/2022] [Indexed: 11/13/2022] Open
Abstract
Viral hepatitis is a leading cause of liver disease and mortality. Infection can occur acutely or chronically, but the mechanisms that govern the clearance of virus or lack thereof are poorly understood and merit further investigation. Though cures for viral hepatitis have been developed, they are expensive, not readily accessible in vulnerable populations and some patients may remain at an increased risk of developing hepatocellular carcinoma (HCC) even after viral clearance. To sustain infection in vitro, hepatocytes must be fully mature and remain in a differentiated state. However, primary hepatocytes rapidly dedifferentiate in conventional 2D in vitro platforms. Physiologically relevant or physiomimetic microsystems, are increasingly popular alternatives to traditional two-dimensional (2D) monocultures for in vitro studies. Physiomimetic systems reconstruct and incorporate elements of the native cellular microenvironment to improve biologic functionality in vitro. Multiple elements contribute to these models including ancillary tissue architecture, cell co-cultures, matrix proteins, chemical gradients and mechanical forces that contribute to increased viability, longevity and physiologic function for the tissue of interest. These microsystems are used in a wide variety of applications to study biological phenomena. Here, we explore the use of physiomimetic microsystems as tools for studying viral hepatitis infection in the liver and how the design of these platforms is tailored for enhanced investigation of the viral lifecycle when compared to conventional 2D cell culture models. Although liver-based physiomimetic microsystems are typically applied in the context of drug studies, the platforms developed for drug discovery purposes offer a solid foundation to support studies on viral hepatitis. Physiomimetic platforms may help prolong hepatocyte functionality in order to sustain chronic viral hepatitis infection in vitro for studying virus-host interactions for prolonged periods.
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Affiliation(s)
- Dennis McDuffie
- Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
| | - David Barr
- Department of Pathology and Laboratory Medicine, University of Miami Miller School of Medicine, Miami, FL, United States
| | - Ashutosh Agarwal
- Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
- Desai Sethi Urology Institute, University of Miami Miller School of Medicine, Miami, FL, United States
- Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, United States
| | - Emmanuel Thomas
- Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
- Department of Pathology and Laboratory Medicine, University of Miami Miller School of Medicine, Miami, FL, United States
- Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, United States
- Schiff Center for Liver Diseases, University of Miami Miller School of Medicine, Miami, FL, United States
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20
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Panahi F, Baheiraei N, Sistani MN, Salehnia M. Analysis of decellularized mouse liver fragment and its recellularization with human endometrial mesenchymal cells as a candidate for clinical usage. Prog Biomater 2022; 11:409-420. [PMID: 36117225 DOI: 10.1007/s40204-022-00203-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Accepted: 09/03/2022] [Indexed: 11/27/2022] Open
Abstract
Decellularized tissue has been used as a natural extracellular matrix (ECM) or bioactive biomaterial for tissue engineering. The present study aims to compare and analyze different decellularization protocols for mouse liver fragments and cell seeding and attachment in the created scaffold using human endometrial mesenchymal cells (hEMCs).After collecting and dissecting the mouse liver into small fragments, they were decellularized by Triton X-100 and six concentrations of sodium dodecyl sulfate (SDS; 0.025, 0.05, 0.1, 0.25, 0.5, and 1%) at different exposure times. The morphology and DNA content of decellularized tissues were studied, and the group with better morphology and lower DNA content was selected for additional assessments. Masson's tri-chrome and periodic acid Schiff staining were performed to evaluate ECM materials. Raman confocal spectroscopy analysis was used to quantify the amount of collagen type I, III and IV, glycosaminoglycans and elastin. Scanning electron microscopy and MTT assay were applied to assess the ultrastructure and porosity and cytotoxicity of decellularized scaffolds, respectively. In the final step, hEMCs were seeded on the decellularized scaffold and cultured for one week, and finally the cell attachment and homing were studied morphologically.The treated group with 0.1% SDS for 24 h showed a well preserved ECM morphology similar to native control and showing the minimum level of DNA. Raman spectroscopy results demonstrated that the amount of collagen type I and IV was not significantly changed in this group compared to the control, but a significant reduction in collagen III and elastin protein levels was seen (P < 0.001). The micrographs showed a porous ECM in decellularized sample similar to the native control with the range of 2.25 µm to 7.86 µm. After cell seeding, the infiltration and migration of cells in different areas of the scaffold were seen. In conclusion, this combined protocol for mouse liver decellularization is effective and its recellularization with hEMCs could be suitable for clinical applications in the future.
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Affiliation(s)
- Fatomeh Panahi
- Department of Biomaterial Engineering, Faculty of Interdisciplinary Sciences and Technologies, Tarbiat Modares University, Tehran, Iran
| | - Nafiseh Baheiraei
- Tissue Engineering Division, Anatomy Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Maryam Nezhad Sistani
- Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, P. O. BOX: 14115-111, Tehran, Iran
| | - Mojdeh Salehnia
- Department of Biomaterial Engineering, Faculty of Interdisciplinary Sciences and Technologies, Tarbiat Modares University, Tehran, Iran. .,Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, P. O. BOX: 14115-111, Tehran, Iran.
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3D Bioprinting of Multi-Material Decellularized Liver Matrix Hydrogel at Physiological Temperatures. BIOSENSORS 2022; 12:bios12070521. [PMID: 35884324 PMCID: PMC9313433 DOI: 10.3390/bios12070521] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Revised: 06/13/2022] [Accepted: 07/08/2022] [Indexed: 12/15/2022]
Abstract
Bioprinting is an acclaimed technique that allows the scaling of 3D architectures in an organized pattern but suffers from a scarcity of appropriate bioinks. Decellularized extracellular matrix (dECM) from xenogeneic species has garnered support as a biomaterial to promote tissue-specific regeneration and repair. The prospect of developing dECM-based 3D artificial tissue is impeded by its inherent low mechanical properties. In recent years, 3D bioprinting of dECM-based bioinks modified with additional scaffolds has advanced the development of load-bearing constructs. However, previous attempts using dECM were limited to low-temperature bioprinting, which is not favorable for a longer print duration with cells. Here, we report the development of a multi-material decellularized liver matrix (dLM) bioink reinforced with gelatin and polyethylene glycol to improve rheology, extrudability, and mechanical stability. This shear-thinning bioink facilitated extrusion-based bioprinting at 37 °C with HepG2 cells into a 3D grid structure with a further enhancement for long-term applications by enzymatic crosslinking with mushroom tyrosinase. The heavily crosslinked structure showed a 16-fold increase in viscosity (2.73 Pa s−1) and a 32-fold increase in storage modulus from the non-crosslinked dLM while retaining high cell viability (85–93%) and liver-specific functions. Our results show that the cytocompatible crosslinking of dLM bioink at physiological temperatures has promising applications for extended 3D-printing procedures.
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22
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Natural Scaffolds Used for Liver Regeneration: A Narrative Update. Stem Cell Rev Rep 2022; 18:2262-2278. [PMID: 35320512 DOI: 10.1007/s12015-022-10362-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/05/2022] [Indexed: 10/18/2022]
Abstract
Annually chronic liver diseases cause two million death worldwide. Although liver transplantation (LT) is still considered the best therapeutic option, the limited number of donated livers and lifelong side effects of LT has led researchers to seek alternative therapies. Tissue engineering (TE) as a promising method is considered for liver repair and regeneration. TE uses natural or synthetic scaffolds, functional somatic cells, multipotent stem cells, and growth factors to develop new organs. Biological scaffolds are notable in TE because of their capacity to mimic extracellular matrices, biodegradability, and biocompatibility. Moreover, natural scaffolds are classified based on their source and function in three separate groups. Hemostat-based scaffolds as the first group were reviewed for their application in coagulation in liver injury or surgery. Furthermore, recent studies showed improvement in the function of biological hydrogels in liver regeneration and vascularity. In addition, different applications of natural scaffolds were discussed and compared with synthetic scaffolds. Finally, we focused on the efforts to improve the performance of decellularized extracellular matrixes for liver implantation.
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Ergun C, Parmaksiz M, Vurat MT, Elçin AE, Elçin YM. Decellularized liver ECM-based 3D scaffolds: Compositional, physical, chemical, rheological, thermal, mechanical, and in vitro biological evaluations. Int J Biol Macromol 2022; 200:110-123. [PMID: 34971643 DOI: 10.1016/j.ijbiomac.2021.12.086] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2021] [Revised: 12/01/2021] [Accepted: 12/15/2021] [Indexed: 12/18/2022]
Abstract
The extracellular matrix (ECM) is involved in many critical cellular interactions through its biological macromolecules. In this study, a macroporous 3D scaffold originating from decellularized bovine liver ECM (dL-ECM), with defined compositional, physical, chemical, rheological, thermal, mechanical, and in vitro biological properties was developed. First, protocols were determined that effectively remove cells and DNA while ECM retains biological macromolecules collagen, elastin, sGAGs in tissue. Rheological analysis revealed the elastic properties of pepsin-digested dL-ECM. Then, dL-ECM hydrogel was neutralized, molded, formed into macroporous (~100-200 μm) scaffolds in aqueous medium at 37 °C, and lyophilized. The scaffolds had water retention ability, and were mechanically stable for at least 14 days in the culture medium. The findings also showed that increasing the dL-ECM concentration from 10 mg/mL to 20 mg/mL resulted in a significant increase in the mechanical strength of the scaffolds. The hemolysis test revealed high in vitro hemocompatibility of the dL-ECM scaffolds. Studies investigating the viability and proliferation status of human adipose stem cells seeded over a 2-week culture period have demonstrated the suitability of dL-ECM scaffolds as a cell substrate. Prospective studies may reveal the extent to which 3D dL-ECM sponges have the potential to create a biomimetic environment for cells.
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Affiliation(s)
- Can Ergun
- Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Stem Cell Institute, Ankara, Turkey
| | - Mahmut Parmaksiz
- Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Stem Cell Institute, Ankara, Turkey
| | - Murat Taner Vurat
- Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Stem Cell Institute, Ankara, Turkey
| | - Ayşe Eser Elçin
- Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Stem Cell Institute, Ankara, Turkey
| | - Yaşar Murat Elçin
- Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Stem Cell Institute, Ankara, Turkey; Biovalda Health Technologies, Inc., Ankara, Turkey.
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24
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Dai Q, Jiang W, Huang F, Song F, Zhang J, Zhao H. Recent Advances in Liver Engineering With Decellularized Scaffold. Front Bioeng Biotechnol 2022; 10:831477. [PMID: 35223793 PMCID: PMC8866951 DOI: 10.3389/fbioe.2022.831477] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Accepted: 01/24/2022] [Indexed: 12/02/2022] Open
Abstract
Liver transplantation is currently the only effective treatment for patients with end-stage liver disease; however, donor liver scarcity is a notable concern. As a result, extensive endeavors have been made to diversify the source of donor livers. For example, the use of a decellularized scaffold in liver engineering has gained considerable attention in recent years. The decellularized scaffold preserves the original orchestral structure and bioactive chemicals of the liver, and has the potential to create a de novo liver that is fit for transplantation after recellularization. The structure of the liver and hepatic extracellular matrix, decellularization, recellularization, and recent developments are discussed in this review. Additionally, the criteria for assessment and major obstacles in using a decellularized scaffold are covered in detail.
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Affiliation(s)
- Qingqing Dai
- Department of Hepatopancreatobiliary Surgery and Organ Transplantation Center, Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China
- Department of Internal Medicine IV (Gastroenterology, Hepatology, and Infectious Diseases), Jena University Hospital, Jena, Germany
| | - Wei Jiang
- Department of Burns, The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Fan Huang
- Department of Hepatopancreatobiliary Surgery and Organ Transplantation Center, Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Fei Song
- Department of Urology, Jena University Hospital, Jena, Germany
| | - Jiqian Zhang
- Department of Anesthesiology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
- *Correspondence: Jiqian Zhang, ; Hongchuan Zhao,
| | - Hongchuan Zhao
- Department of Hepatopancreatobiliary Surgery and Organ Transplantation Center, Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China
- *Correspondence: Jiqian Zhang, ; Hongchuan Zhao,
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25
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Yadav S, Majumder A. Biomimicked hierarchical 2D and 3D structures from natural templates: applications in cell biology. Biomed Mater 2021; 16. [PMID: 34438385 DOI: 10.1088/1748-605x/ac21a7] [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: 01/02/2021] [Accepted: 08/26/2021] [Indexed: 11/11/2022]
Abstract
Intricate structures of natural surfaces and materials have amazed people over the ages. The unique properties of various surfaces also created interest and curiosity in researchers. In the recent past, with the advent of superior microscopy techniques, we have started to understand how these complex structures provide superior properties. With that knowledge, scientists have developed various biomimicked and bio-inspired surfaces for different non-biological applications. In the last two decades, we have also started to learn how structures of the tissue microenvironment influence cell function and behaviour, both in physiological and pathological conditions. Hence, it became essential to decipher the role and importance of structural hierarchy in the cellular context. With advances in microfabricated techniques, such complex structures were made by superimposing features of different dimensions. However, the fabricated topographies are far from matching the complexities presentin vivo. Hence, the need of biomimicking the natural surfaces for cellular applications was felt. In this review, we discuss a few examples of hierarchical surfaces found in plants, insects, and vertebrates. Such structures have been widely biomimicked for various applications but rarely studied for cell-substrate interaction and cellular response. Here, we discuss the research work wherein 2D hierarchical substrates were prepared using biomimicking to understand cellular functions such as adhesion, orientation, differentiation, and formation of spheroids. Further, we also present the status of ongoing research in mimicking 3D tissue architecture using de-cellularized plant-based and tissue/organ-based scaffolds. We will also discuss 3D printing for fabricating 2D and 3D hierarchical structures. The review will end by highlighting the various advantages and research challenges in this approach. The biomimickedin-vivolike substrate can be used to better understand cellular physiology, and for tissue engineering.
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Affiliation(s)
- Shital Yadav
- Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai 400076, India
| | - Abhijit Majumder
- Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai 400076, India
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26
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Characterization of Indonesia Decellularized Liver Cubes Scaffold using Scanning Electron Microscopy. JOURNAL OF BIOMIMETICS BIOMATERIALS AND BIOMEDICAL ENGINEERING 2021. [DOI: 10.4028/www.scientific.net/jbbbe.52.38] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Liver biological scaffold was developed in order to resemble native liver tissue environment. It can be achieved by decellularizing native liver tissue that will remove cells and preserve extracellular matrix (ECM). Furthermore, ECM fibers are arranged in a special pattern, which affect liver cell polarity and topography that are important for cells’ implantation, proliferation and differentiation. Therefore, the aim of this study was to evaluate liver cube scaffold topography that was decellularized with fixed multiple sites syringe injection (Indonesia patent number: S00201907930).Rat liver cubes (n=3) underwent decellularization with Ethylene Glycol Tetraacetic Acid (EGTA) immersion and increased Sodium Dodecyl Sulfate (SDS) concentrations using previous multiple sites syringe injection protocol study. Deoxyribonucleic Acid (DNA) concentrations were measured to confirm less DNA materials remaining in scaffolds. Scanning Electron Microscope (SEM) analysis of scaffolds were conducted for topographic characterization compared to undecellularized liver control. Molecular analysis of DNA concentration showed complete removal of DNA material. SEM analysis gave appearance of intact liver cube scaffold microarchitecture. Liver cubes decellularization using multiple sites syringe injection showed good topographic liver scaffold characterization.
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27
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Khajavi M, Hashemi M, Kalalinia F. Recent advances in optimization of liver decellularization procedures used for liver regeneration. Life Sci 2021; 281:119801. [PMID: 34229008 DOI: 10.1016/j.lfs.2021.119801] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2021] [Revised: 06/19/2021] [Accepted: 06/29/2021] [Indexed: 10/20/2022]
Abstract
Severe liver diseases have been considered the most common causes of adult deaths worldwide. Until now, liver transplantation is known as the only effective treatment for end stage liver disease. However, it is associated with several problems, most importantly, the side effects of immunosuppressive drugs that should be used after transplantation, and the shortage of tissue donors compared to the increasing number of patients requiring liver transplantation. Currently, tissue/organ decellularization as a new approach in tissue engineering is becoming a valid substitute for managing these kinds of problems. Decellularization of a whole liver is an attractive procedure to create three-dimensional (3D) scaffolds that micro-architecturally and structurally are similar to the native one and could support the repair or replacement of damaged or injured tissue. In this review, the different methods used for decellularization of liver tissue have been reviewed. In addition, the current approaches to overcome the challenges in these techniques are discussed.
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Affiliation(s)
- Mohaddeseh Khajavi
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Maryam Hashemi
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Fatemeh Kalalinia
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
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28
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Behmer Hansen RA, Wang X, Kaw G, Pierre V, Senyo SE. Accounting for Material Changes in Decellularized Tissue with Underutilized Methodologies. BIOMED RESEARCH INTERNATIONAL 2021; 2021:6696295. [PMID: 34159202 PMCID: PMC8187050 DOI: 10.1155/2021/6696295] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 05/05/2021] [Accepted: 05/21/2021] [Indexed: 11/17/2022]
Abstract
Tissue decellularization has rapidly developed to be a practical approach in tissue engineering research; biological tissue is cleared of cells resulting in a protein-rich husk as a natural scaffold for growing transplanted cells as a donor organ therapy. Minimally processed, acellular extracellular matrix reproduces natural interactions with cells in vitro and for tissue engineering applications in animal models. There are many decellularization techniques that achieve preservation of molecular profile (proteins and sugars), microstructure features such as organization of ECM layers (interstitial matrix and basement membrane) and organ level macrofeatures (vasculature and tissue compartments). While structural and molecular cues receive attention, mechanical and material properties of decellularized tissues are not often discussed. The effects of decellularization on an organ depend on the tissue properties, clearing mechanism, chemical interactions, solubility, temperature, and treatment duration. Physical characterization by a few labs including work from the authors provides evidence that decellularization protocols should be tailored to specific research questions. Physical characterization beyond histology and immunohistochemistry of the decellularized matrix (dECM) extends evaluation of retained functional features of the original tissue. We direct our attention to current technologies that can be employed for structure function analysis of dECM using underutilized tools such as atomic force microscopy (AFM), cryogenic electron microscopy (cryo-EM), dynamic mechanical analysis (DMA), Fourier-transform infrared spectroscopy (FTIR), mass spectrometry, and rheometry. Structural imaging and mechanical functional testing combined with high-throughput molecular analyses opens a new approach for a deeper appreciation of how cellular behavior is influenced by the isolated microenvironment (specifically dECM). Additionally, the impact of these features with different decellularization techniques and generation of synthetic material scaffolds with desired attributes are informed. Ultimately, this mechanical profiling provides a new dimension to our understanding of decellularized matrix and its role in new applications.
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Affiliation(s)
- Ryan A. Behmer Hansen
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
| | - Xinming Wang
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
| | - Gitanjali Kaw
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
| | - Valinteshley Pierre
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
| | - Samuel E. Senyo
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
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29
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Massaro MS, Pálek R, Rosendorf J, Červenková L, Liška V, Moulisová V. Decellularized xenogeneic scaffolds in transplantation and tissue engineering: Immunogenicity versus positive cell stimulation. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2021; 127:112203. [PMID: 34225855 DOI: 10.1016/j.msec.2021.112203] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 05/13/2021] [Accepted: 05/18/2021] [Indexed: 01/22/2023]
Abstract
Seriously compromised function of some organs can only be restored by transplantation. Due to the shortage of human donors, the need to find another source of organs is of primary importance. Decellularized scaffolds of non-human origin are being studied as highly potential biomaterials for tissue engineering. Their biological nature and thus the ability to provide a naturally-derived environment for human cells to adhere and grow highlights their great advantage in comparison to synthetic scaffolds. Nevertheless, since every biomaterial implanted in the body generates immune reaction, studying the interaction of the scaffold with the surrounding tissues is necessary. This review aims to summarize current knowledge on the immunogenicity of semi-xenografts involved in transplantation. Moreover, positive aspects of the interaction between xenogeneic scaffold and human cells are discussed, focusing on specific roles of proteins associated with extracellular matrix in cell adhesion and signalling.
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Affiliation(s)
- Maria Stefania Massaro
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 32300 Pilsen, Czech Republic
| | - Richard Pálek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 32300 Pilsen, Czech Republic; Department of Surgery, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 80, 32300 Pilsen, Czech Republic
| | - Jáchym Rosendorf
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 32300 Pilsen, Czech Republic; Department of Surgery, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 80, 32300 Pilsen, Czech Republic
| | - Lenka Červenková
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 32300 Pilsen, Czech Republic; Department of Pathology, Third Faculty of Medicine, Charles University, Ruska 87, 100 00 Prague 10, Czech Republic
| | - Václav Liška
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 32300 Pilsen, Czech Republic; Department of Surgery, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 80, 32300 Pilsen, Czech Republic
| | - Vladimíra Moulisová
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 32300 Pilsen, Czech Republic.
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30
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Asadi M, Khalili M, Lotfi H, Vaghefi Moghaddam S, Zarghami N, André H, Alizadeh E. Liver bioengineering: Recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic. Life Sci 2021; 276:119373. [PMID: 33744324 DOI: 10.1016/j.lfs.2021.119373] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2021] [Revised: 03/03/2021] [Accepted: 03/08/2021] [Indexed: 02/07/2023]
Abstract
Development of novel technologies provides the best tissue constructs engineering and maximizes their therapeutic effects in regenerative therapy, especially for liver dysfunctions. Among the currently investigated approaches of tissue engineering, scaffold-based and scaffold-free tissues are widely suggested for liver regeneration. Analogs of liver acellular extracellular matrix (ECM) are utilized in native scaffolds to increase the self-repair and healing ability of organs. Native ECM analog could improve liver repairing through providing the supportive framework for cells and signaling molecules, exerting normal biomechanical, biochemical, and physiological signal complexes. Recently, innovative cell sheet technology is introduced as an alternative for conventional tissue engineering with the advantage of fewer scaffold restrictions and cell culture on a Thermo-Responsive Polymer Surface. These sheets release the layered cells through a temperature-controlled procedure without enzymatic digestion, while preserving the cell-ECM contacts and adhesive molecules on cell-cell junctions. In addition, several novelties have been introduced into the cell sheet and decellularization technologies to aid cell growth, instruct differentiation/angiogenesis, and promote cell migration. In this review, recent trends, advancements, and issues linked to translation into clinical practice are dissected and compared regarding the decellularization and cell sheet technologies for liver tissue engineering.
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Affiliation(s)
- Maryam Asadi
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mostafa Khalili
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hajie Lotfi
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Physiology, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Nosratollah Zarghami
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Helder André
- Department of Clinical Neuroscience, St. Erik Eye Hospital, Karolinska Institute, 11282 Stockholm, Sweden
| | - Effat Alizadeh
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
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31
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Capella-Monsonís H, Zeugolis DI. Decellularized xenografts in regenerative medicine: From processing to clinical application. Xenotransplantation 2021; 28:e12683. [PMID: 33709410 DOI: 10.1111/xen.12683] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2020] [Revised: 01/28/2021] [Accepted: 02/25/2021] [Indexed: 12/13/2022]
Abstract
Decellularized xenografts are an inherent component of regenerative medicine. Their preserved structure, mechanical integrity and biofunctional composition have well established them in reparative medicine for a diverse range of clinical indications. Nonetheless, their performance is highly influenced by their source (ie species, age, tissue) and processing (ie decellularization, crosslinking, sterilization and preservation), which govern their final characteristics and determine their success or failure for a specific clinical target. In this review, we provide an overview of the different sources and processing methods used in decellularized xenografts fabrication and discuss their effect on the clinical performance of commercially available decellularized xenografts.
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Affiliation(s)
- Héctor Capella-Monsonís
- 1Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland.,Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland
| | - Dimitrios I Zeugolis
- 1Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland.,Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland.,Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Faculty of Biomedical Sciences, Università della Svizzera Italiana (USI), Lugano, Switzerland
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32
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A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs. NANOMATERIALS 2021; 11:nano11020275. [PMID: 33494337 PMCID: PMC7912543 DOI: 10.3390/nano11020275] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 01/11/2021] [Accepted: 01/19/2021] [Indexed: 02/06/2023]
Abstract
In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.
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Lorvellec M, Pellegata AF, Maestri A, Turchetta C, Alvarez Mediavilla E, Shibuya S, Jones B, Scottoni F, Perocheau DP, Cozmescu AC, Delhove JM, Kysh D, Gjinovci A, Counsell JR, Heywood WE, Mills K, McKay TR, De Coppi P, Gissen P. An In Vitro Whole-Organ Liver Engineering for Testing of Genetic Therapies. iScience 2020; 23:101808. [PMID: 33305175 PMCID: PMC7708813 DOI: 10.1016/j.isci.2020.101808] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 10/19/2020] [Accepted: 11/10/2020] [Indexed: 12/30/2022] Open
Abstract
Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.
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Affiliation(s)
- Maëlle Lorvellec
- MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Alessandro Filippo Pellegata
- Developmental Biology and Cancer Research & Teaching Department, Stem Cells & Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Alice Maestri
- MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Chiara Turchetta
- Department of Chemistry, Materials and Chemical Engineering "Giulio Natta," Politecnico di Milano, Milan 20133, Italy
| | - Elena Alvarez Mediavilla
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Soichi Shibuya
- Developmental Biology and Cancer Research & Teaching Department, Stem Cells & Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Brendan Jones
- Developmental Biology and Cancer Research & Teaching Department, Stem Cells & Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Federico Scottoni
- Developmental Biology and Cancer Research & Teaching Department, Stem Cells & Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Dany P. Perocheau
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Andrei Claudiu Cozmescu
- MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
- NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London, London WC1N 1EH, UK
| | - Juliette M. Delhove
- Robinson Research Institute, University of Adelaide, Adelaide, SA, 5006, Australia
| | - Daniel Kysh
- MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
| | - Asllan Gjinovci
- MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
| | - John R. Counsell
- Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Programme, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK
| | - Wendy E. Heywood
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Kevin Mills
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Tristan R. McKay
- Centre for Bioscience, Manchester Metropolitan University, Manchester M1 5GD, UK
| | - Paolo De Coppi
- Developmental Biology and Cancer Research & Teaching Department, Stem Cells & Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
| | - Paul Gissen
- MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK
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Jirik M, Gruber I, Moulisova V, Schindler C, Cervenkova L, Palek R, Rosendorf J, Arlt J, Bolek L, Dejmek J, Dahmen U, Zelezny M, Liska V. Semantic Segmentation of Intralobular and Extralobular Tissue from Liver Scaffold H&E Images. SENSORS 2020; 20:s20247063. [PMID: 33321713 PMCID: PMC7764590 DOI: 10.3390/s20247063] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 12/02/2020] [Accepted: 12/07/2020] [Indexed: 12/13/2022]
Abstract
Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.
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Affiliation(s)
- Miroslav Jirik
- NTIS—New Technologies for the Information Society, Faculty of Applied Sciences, University of West Bohemia, 301 00 Pilsen, Czech Republic; (I.G.); (M.Z.)
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
- Correspondence:
| | - Ivan Gruber
- NTIS—New Technologies for the Information Society, Faculty of Applied Sciences, University of West Bohemia, 301 00 Pilsen, Czech Republic; (I.G.); (M.Z.)
| | - Vladimira Moulisova
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
| | - Claudia Schindler
- Experimental Transplantation Surgery Department, Universitätsklinikum Jena, 07743 Jena, Germany; (C.S.); (J.A.); (U.D.)
| | - Lenka Cervenkova
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
| | - Richard Palek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
- Department of Surgery, University Hospital and Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic
| | - Jachym Rosendorf
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
- Department of Surgery, University Hospital and Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic
| | - Janine Arlt
- Experimental Transplantation Surgery Department, Universitätsklinikum Jena, 07743 Jena, Germany; (C.S.); (J.A.); (U.D.)
| | - Lukas Bolek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
| | - Jiri Dejmek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
| | - Uta Dahmen
- Experimental Transplantation Surgery Department, Universitätsklinikum Jena, 07743 Jena, Germany; (C.S.); (J.A.); (U.D.)
| | - Milos Zelezny
- NTIS—New Technologies for the Information Society, Faculty of Applied Sciences, University of West Bohemia, 301 00 Pilsen, Czech Republic; (I.G.); (M.Z.)
| | - Vaclav Liska
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic; (V.M.); (L.C.); (R.P.); (J.R.); (L.B.); (J.D.); (V.L.)
- Department of Surgery, University Hospital and Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic
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Abaci A, Guvendiren M. Designing Decellularized Extracellular Matrix-Based Bioinks for 3D Bioprinting. Adv Healthc Mater 2020; 9:e2000734. [PMID: 32691980 DOI: 10.1002/adhm.202000734] [Citation(s) in RCA: 95] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/10/2020] [Indexed: 12/17/2022]
Abstract
3D bioprinting is an emerging technology to fabricate tissues and organs by precisely positioning cells into 3D structures using printable cell-laden formulations known as bioinks. Various bioinks are utilized in 3D bioprinting applications; however, developing the perfect bioink to fabricate constructs with biomimetic microenvironment and mechanical properties that are similar to native tissues is a challenging task. In recent years, decellularized extracellular matrix (dECM)-based bioinks have received an increasing attention in 3D bioprinting applications, since they are derived from native tissues and possess unique, complex tissue-specific biochemical properties. This review focuses on designing dECM-based bioinks for tissue and organ bioprinting, including commonly used decellularization and decellularized tissue characterization methods, bioink formulation and characterization, applications of dECM-based bioinks, and most recent advancements in dECM-based bioink design.
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Affiliation(s)
- Alperen Abaci
- Instructive Biomaterials and Additive Manufacturing Laboratory Otto H. York Chemical and Materials Engineering 138 York Center New Jersey Institute of Technology University Heights Newark NJ 07102 USA
| | - Murat Guvendiren
- Instructive Biomaterials and Additive Manufacturing Laboratory Otto H. York Chemical and Materials Engineering 138 York Center New Jersey Institute of Technology University Heights Newark NJ 07102 USA
- Department of Biomedical Engineering New Jersey Institute of Technology University Heights Newark NJ 07102 USA
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36
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Papatheodoridi M, Mazza G, Pinzani M. Regenerative hepatology: In the quest for a modern prometheus? Dig Liver Dis 2020; 52:1106-1114. [PMID: 32868215 DOI: 10.1016/j.dld.2020.08.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/19/2020] [Revised: 07/30/2020] [Accepted: 08/03/2020] [Indexed: 12/11/2022]
Abstract
As liver-related morbidity and mortality is rising worldwide and orthotopic liver transplantation (OLT) remains the only standard-of-care for end-stage liver disease or acute liver failure, shortage of donor organs is becoming more prominent. Importantly, advances in regenerative Hepatology and liver bioengineering are bringing new hope to the possibility of restoring impaired hepatic functionality in the presence of acute or chronic liver failure. Hepatocyte transplantation and artificial liver-support systems were the first strategies used in regenerative hepatology but have presented various types of efficiency limitations restricting their widespread use. In parallel, liver bioengineering has been a rapidly developing field bringing continuously novel advancements in biomaterials, three dimensional (3D) scaffolds, cell sources and relative methodologies for creating bioengineered liver tissue. The current major task in liver bioengineering is to build small implantable liver mass for treating inherited metabolic disorders, bioengineered bile ducts for congenital biliary defects and large bioengineered liver organs for transplantation, as substitutes to donor-organs, in cases of acute or acute-on-chronic liver failure. This review aims to summarize the state-of-the-art and upcoming technologies of regenerative Hepatology that are emerging as promising alternatives to the current standard-of care in liver disease.
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Affiliation(s)
- Margarita Papatheodoridi
- Sheila Sherlock Liver Unit, Institute for Liver and Digestive Health, University College London, London, United Kingdom
| | - Giuseppe Mazza
- Sheila Sherlock Liver Unit, Institute for Liver and Digestive Health, University College London, London, United Kingdom
| | - Massimo Pinzani
- Sheila Sherlock Liver Unit, Institute for Liver and Digestive Health, University College London, London, United Kingdom.
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37
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Moulisová V, Jiřík M, Schindler C, Červenková L, Pálek R, Rosendorf J, Arlt J, Bolek L, Šůsová S, Nietzsche S, Liška V, Dahmen U. Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds. J Tissue Eng 2020; 11:2041731420921121. [PMID: 32523667 PMCID: PMC7257850 DOI: 10.1177/2041731420921121] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Accepted: 04/02/2020] [Indexed: 12/19/2022] Open
Abstract
Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.
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Affiliation(s)
- Vladimíra Moulisová
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Miroslav Jiřík
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Cybernetics, University of West Bohemia, Pilsen, Czech Republic
| | - Claudia Schindler
- Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany
| | - Lenka Červenková
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Pathology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
| | - Richard Pálek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Jáchym Rosendorf
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Janine Arlt
- Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany
| | - Lukáš Bolek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Biophysics, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Simona Šůsová
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Toxicogenomics, National Institute of Public Health, Prague, Czech Republic
| | - Sandor Nietzsche
- Centre for Electron Microscopy, University Hospital Jena, Jena, Germany
| | - Václav Liška
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Uta Dahmen
- Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany
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38
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Decellularized liver matrix as substrates for rescue of acute hepatocytes toxicity. J Biomed Mater Res B Appl Biomater 2020; 108:1592-1602. [DOI: 10.1002/jbm.b.34506] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 08/16/2019] [Accepted: 09/22/2019] [Indexed: 12/11/2022]
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39
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Crowley C, Butler CR, Camilli C, Hynds RE, Kolluri KK, Janes SM, De Coppi P, Urbani L. Non-Invasive Longitudinal Bioluminescence Imaging of Human Mesoangioblasts in Bioengineered Esophagi. Tissue Eng Part C Methods 2020; 25:103-113. [PMID: 30648471 PMCID: PMC6389770 DOI: 10.1089/ten.tec.2018.0351] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Esophageal engineering aims to create replacement solutions by generating hollow organs using a combination of cells, scaffolds, and regeneration-stimulating factors. Currently, the fate of cells on tissue-engineered grafts is generally determined retrospectively by histological analyses. Unfortunately, quality-controlled cell seeding protocols for application in human patients are not standard practice. As such, the field requires simple, fast, and reliable techniques for non-invasive, highly specific cell tracking. Here, we show that bioluminescence imaging (BLI) is a suitable method to track human mesoangioblast seeding of an esophageal tubular construct at every stage of the preclinical bioengineering pipeline. In particular, validation of BLI as longitudinal quantitative assessment of cell density, proliferation, seeding efficiency, bioreactor culture, and cell survival upon implantation in vivo was performed against standard methods in 2D cultures and in 3D decellularized esophageal scaffolds. The technique is simple, non-invasive, and provides information on mesoangioblast distribution over entire scaffolds. Bioluminescence is an invaluable tool in the development of complex bioartificial organs and can assist in the development of standardized cell seeding protocols, with the ability to track cells from bioreactor through to implantation.
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Affiliation(s)
- Claire Crowley
- 1 Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Children's Hospital, University College London, London, United Kingdom
| | - Colin R Butler
- 1 Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Children's Hospital, University College London, London, United Kingdom.,2 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom
| | - Carlotta Camilli
- 1 Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Children's Hospital, University College London, London, United Kingdom
| | - Robert E Hynds
- 2 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom
| | - Krishna K Kolluri
- 2 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom
| | - Sam M Janes
- 2 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom
| | - Paolo De Coppi
- 1 Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Children's Hospital, University College London, London, United Kingdom
| | - Luca Urbani
- 1 Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Children's Hospital, University College London, London, United Kingdom.,3 Institute of Hepatology London, Foundation for Liver Research, London, United Kingdom
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40
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Li Y, Wu Q, Wang Y, Bu H, Bao J. Porcine Hepatocytes: Isolation and Liver Tissue Engineering for Xenotransplantation. Xenotransplantation 2020; 2110:267-287. [DOI: 10.1007/978-1-0716-0255-3_18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/09/2023]
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41
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Hosseini V, Maroufi NF, Saghati S, Asadi N, Darabi M, Ahmad SNS, Hosseinkhani H, Rahbarghazi R. Current progress in hepatic tissue regeneration by tissue engineering. J Transl Med 2019; 17:383. [PMID: 31752920 PMCID: PMC6873477 DOI: 10.1186/s12967-019-02137-6] [Citation(s) in RCA: 64] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2019] [Accepted: 11/12/2019] [Indexed: 12/12/2022] Open
Abstract
Liver, as a vital organ, is responsible for a wide range of biological functions to maintain homeostasis and any type of damages to hepatic tissue contributes to disease progression and death. Viral infection, trauma, carcinoma, alcohol misuse and inborn errors of metabolism are common causes of liver diseases are a severe known reason for leading to end-stage liver disease or liver failure. In either way, liver transplantation is the only treatment option which is, however, hampered by the increasing scarcity of organ donor. Over the past years, considerable efforts have been directed toward liver regeneration aiming at developing new approaches and methodologies to enhance the transplantation process. These approaches include producing decellularized scaffolds from the liver organ, 3D bio-printing system, and nano-based 3D scaffolds to simulate the native liver microenvironment. The application of small molecules and micro-RNAs and genetic manipulation in favor of hepatic differentiation of distinct stem cells could also be exploited. All of these strategies will help to facilitate the application of stem cells in human medicine. This article reviews the most recent strategies to generate a high amount of mature hepatocyte-like cells and updates current knowledge on liver regenerative medicine.
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Affiliation(s)
- Vahid Hosseini
- Stem Cell Research Center, Tabriz University of Medical Sciences, Imam Reza St., Golgasht St., Tabriz, 5166614756, Iran.,Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Nazila Fathi Maroufi
- Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.,Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Sepideh Saghati
- Department of Tissue Engineering, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Nahideh Asadi
- Department of Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Masoud Darabi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Imam Reza St., Golgasht St., Tabriz, 5166614756, Iran.,Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Saeed Nazari Soltan Ahmad
- Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Reza Rahbarghazi
- Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
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42
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Ventura RD, Padalhin AR, Park CM, Lee BT. Enhanced decellularization technique of porcine dermal ECM for tissue engineering applications. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2019; 104:109841. [DOI: 10.1016/j.msec.2019.109841] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 02/22/2019] [Accepted: 05/30/2019] [Indexed: 01/25/2023]
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43
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Raghunathan R, Sethi MK, Klein JA, Zaia J. Proteomics, Glycomics, and Glycoproteomics of Matrisome Molecules. Mol Cell Proteomics 2019; 18:2138-2148. [PMID: 31471497 PMCID: PMC6823855 DOI: 10.1074/mcp.r119.001543] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2019] [Revised: 08/26/2019] [Indexed: 12/21/2022] Open
Abstract
The most straightforward applications of proteomics database searching involve intracellular proteins. Although intracellular gene products number in the thousands, their well-defined post-translational modifications (PTMs) makes database searching practical. By contrast, cell surface and extracellular matrisome proteins pass through the secretory pathway where many become glycosylated, modulating their physicochemical properties, adhesive interactions, and diversifying their functions. Although matrisome proteins number only a few hundred, their high degree of complex glycosylation multiplies the number of theoretical proteoforms by orders of magnitude. Given that extracellular networks that mediate cell-cell and cell-pathogen interactions in physiology depend on glycosylation, it is important to characterize the proteomes, glycomes, and glycoproteomes of matrisome molecules that exist in a given biological context. In this review, we summarize proteomics approaches for characterizing matrisome molecules, with an emphasis on applications to brain diseases. We demonstrate the availability of methods that should greatly increase the availability of information on matrisome molecular structure associated with health and disease.
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Affiliation(s)
- Rekha Raghunathan
- Molecular and Translational Medicine Program, Boston University, Boston, MA 02218; Department of Biochemistry, Boston University, Boston, MA 02218
| | - Manveen K Sethi
- Department of Biochemistry, Boston University, Boston, MA 02218
| | - Joshua A Klein
- Bioinformatics Program, Boston University, Boston, MA 02218
| | - Joseph Zaia
- Molecular and Translational Medicine Program, Boston University, Boston, MA 02218; Department of Biochemistry, Boston University, Boston, MA 02218; Bioinformatics Program, Boston University, Boston, MA 02218.
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44
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Bizzaro D, Russo FP, Burra P. New Perspectives in Liver Transplantation: From Regeneration to Bioengineering. Bioengineering (Basel) 2019; 6:81. [PMID: 31514475 PMCID: PMC6783848 DOI: 10.3390/bioengineering6030081] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Revised: 09/09/2019] [Accepted: 09/10/2019] [Indexed: 12/18/2022] Open
Abstract
Advanced liver diseases have very high morbidity and mortality due to associated complications, and liver transplantation represents the only current therapeutic option. However, due to worldwide donor shortages, new alternative approaches are mandatory for such patients. Regenerative medicine could be the more appropriate answer to this need. Advances in knowledge of physiology of liver regeneration, stem cells, and 3D scaffolds for tissue engineering have accelerated the race towards efficient therapies for liver failure. In this review, we propose an update on liver regeneration, cell-based regenerative medicine and bioengineering alternatives to liver transplantation.
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Affiliation(s)
- Debora Bizzaro
- Department of Surgery, Oncology and Gastroenterology, Gastroenterology/Multivisceral Transplant Section, University/Hospital Padua, 35128 Padua, Italy.
| | - Francesco Paolo Russo
- Department of Surgery, Oncology and Gastroenterology, Gastroenterology/Multivisceral Transplant Section, University/Hospital Padua, 35128 Padua, Italy.
| | - Patrizia Burra
- Department of Surgery, Oncology and Gastroenterology, Gastroenterology/Multivisceral Transplant Section, University/Hospital Padua, 35128 Padua, Italy.
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Controlled cross‐linking of porcine cholecyst extracellular matrix for preparing tissue engineering scaffold. J Biomed Mater Res B Appl Biomater 2019; 108:1057-1067. [DOI: 10.1002/jbm.b.34457] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2019] [Revised: 06/27/2019] [Accepted: 07/17/2019] [Indexed: 12/31/2022]
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Xu L, Huang Y, Wang D, Zhu S, Wang Z, Yang Y, Guo Y. Reseeding endothelial cells with fibroblasts to improve the re-endothelialization of pancreatic acellular scaffolds. JOURNAL OF MATERIALS SCIENCE. MATERIALS IN MEDICINE 2019; 30:85. [PMID: 31292746 DOI: 10.1007/s10856-019-6287-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/27/2019] [Accepted: 06/29/2019] [Indexed: 06/09/2023]
Abstract
Pancreatic transplantation remains the only cure for diabetes, but the shortage of donors limits its clinical application. Whole organ decellularized scaffolds offer a new opportunity for pancreatic organ regeneration; however inadequate endothelialization and vascularization can prevent sufficient transport of oxygen and nutrient supplies to the transplanted organ, as well as leading unwanted thrombotic events. In the present study, we explored the re-endothelialization of rat pancreatic acellular scaffolds via circulation perfusion using human skin fibroblasts (FBs) and human umbilical vein endothelial cells (HUVECs). Our results revealed that the cell adhesion rate when these cells were co-cultured was higher than under control conditions, and this increase was associated with increased release of growth factors including VEGF, FGFb, EGF, and IGF-1 as measured by ELISA. When these recellularized organs were implanted in vivo for 28 days in rat dorsal subcutaneous pockets, we found that de novo vasculature formation in the co-culture samples was superior to the control samples. Together these results suggest that endothelial cell and FB co-culture enhances the re-endothelialization and vascularization of pancreatic acellular scaffolds.
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Affiliation(s)
- Liancheng Xu
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, China
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, China
| | - Yan Huang
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, China
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, China
| | - Dongzhi Wang
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, China
| | - Shajun Zhu
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Zhiwei Wang
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Yumin Yang
- Key Laboratory of Neuroregeneration, Neural Regeneration Co-Innovation Center of Jiangsu Province, Nantong University, Nantong, China.
| | - Yibing Guo
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, China.
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Advances in Hepatic Tissue Bioengineering with Decellularized Liver Bioscaffold. Stem Cells Int 2019; 2019:2693189. [PMID: 31198426 PMCID: PMC6526559 DOI: 10.1155/2019/2693189] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2018] [Revised: 02/08/2019] [Accepted: 03/17/2019] [Indexed: 12/28/2022] Open
Abstract
The burden of liver diseases continues to grow worldwide, and liver transplantation is the only option for patients with end-stage liver disease. This procedure is limited by critical issues, including the low availability of donor organs; thus, novel therapeutic strategies are greatly needed. Recently, bioengineering approaches using decellularized liver scaffolds have been proposed as a novel strategy to overcome these challenges. The aim of this systematic literature review was to identify the major advances in the field of bioengineering using decellularized liver scaffolds and to identify obstacles and challenges for clinical application. The main findings of the articles and each contribution for technique optimization were highlighted, including the protocols of perfusion and decellularization, duration, demonstration of quality control—scaffold acellularity, matrix composition, and preservation of growth factors—and tissue functionality after recellularization. In previous years, many advances have been made as this technique has evolved from studies in animal models to human livers. As the field develops and this promising technique has become much more feasible, many challenges remain, including the selection of appropriate cell types for recellularization, route of cell administration, cell-seeding protocol, and scalability that must be standardized prior to clinical application.
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Zhang Z, Xu H, Mazza G, Zhang M, Frenguelli L, Liu Q, Al-Akkad W, Ren J, Zhao R, Ren F, Chen X, Huang A, Chen J. Decellularized human liver scaffold-based three-dimensional culture system facilitate hepatitis B virus infection. J Biomed Mater Res A 2019; 107:1744-1753. [PMID: 30963688 DOI: 10.1002/jbm.a.36690] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 02/18/2019] [Accepted: 03/15/2019] [Indexed: 12/21/2022]
Abstract
Hepatitis B virus (HBV) study is hampered by lacking of idea cell model which support effective HBV infection and meanwhile recapitulate hepatocyte biology function in vivo. In this study, we developed decellularized human liver scaffolds for cell culture and further applied for HBV infection. As a result, primary human hepatocytes (PHHs) engrafted into liver scaffolds and maintained differentiation with stable albumin secretion and liver-specific gene expression. Comparing to mono-layer cell culture, scaffold-based three-dimensional (3D) culture system significantly augment HBV DNA (including cccDNA), RNA level as well as HBsAg secretion. Moreover, HepG2-NTCP cells cultured on 3D system exhibited higher infection efficiency and longer infection period in vitro. In addition, HBV DNA level was suppressed when anti-HBV medicine Entecavir (ETV) introduced into HepG2-NTCP 3D system. Herein, we evaluated the potential of decellularized human liver scaffold-based in 3D cell culture and disclosed that scaffold-based 3D culture system can facilitate HBV infection in vitro. This 3D culture system could be further applied in HBV-related study. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1744-1753, 2019.
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Affiliation(s)
- ZhenZhen Zhang
- Ministry of Education Key Laboratory of Child Development and Disorders, ChongQing, China
- Key Laboratory of Pediatrics in Chongqing, Chongqing, China
- Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China
- Department of Infectious Disease, Children's Hospital of ChongQing Medical University, ChongQing, China
| | - HongMei Xu
- Ministry of Education Key Laboratory of Child Development and Disorders, ChongQing, China
- Key Laboratory of Pediatrics in Chongqing, Chongqing, China
- Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China
- Department of Infectious Disease, Children's Hospital of ChongQing Medical University, ChongQing, China
| | - Giuseppe Mazza
- UCL Institute for Liver and Digestive Health, Royal Free Hospital, University College London, London, United Kingdom
| | - MingMan Zhang
- Department of Hepatobiliary Surgery, Children's Hospital of ChongQing Medical University, ChongQing, China
| | - Luca Frenguelli
- UCL Institute for Liver and Digestive Health, Royal Free Hospital, University College London, London, United Kingdom
| | - QuanBo Liu
- Ministry of Education Key Laboratory of Child Development and Disorders, ChongQing, China
- Key Laboratory of Pediatrics in Chongqing, Chongqing, China
- Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China
- Department of Infectious Disease, Children's Hospital of ChongQing Medical University, ChongQing, China
| | - Walid Al-Akkad
- UCL Institute for Liver and Digestive Health, Royal Free Hospital, University College London, London, United Kingdom
| | - JiHua Ren
- The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, ChongQing, China
- Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, ChongQing, 400016, China
| | - RuiQiu Zhao
- Ministry of Education Key Laboratory of Child Development and Disorders, ChongQing, China
- Key Laboratory of Pediatrics in Chongqing, Chongqing, China
- Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China
- Department of Infectious Disease, Children's Hospital of ChongQing Medical University, ChongQing, China
| | - Fang Ren
- The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, ChongQing, China
- Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, ChongQing, 400016, China
| | - Xin Chen
- Ministry of Education Key Laboratory of Child Development and Disorders, ChongQing, China
- Key Laboratory of Pediatrics in Chongqing, Chongqing, China
- Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China
- The General Gard, Children's Hospital of ChongQing Medical University, ChongQing, China
| | - AiLong Huang
- The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, ChongQing, China
- Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, ChongQing, 400016, China
| | - Juan Chen
- The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, ChongQing, China
- Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, ChongQing, 400016, China
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Zambaiti E, Scottoni F, Rizzi E, Russo S, Deguchi K, Eaton S, Pellegata AF, De Coppi P. Whole rat stomach decellularisation using a detergent-enzymatic protocol. Pediatr Surg Int 2019; 35:21-27. [PMID: 30443739 PMCID: PMC6326006 DOI: 10.1007/s00383-018-4372-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 10/18/2018] [Indexed: 02/03/2023]
Abstract
BACKGROUND Conditions leading to reduced gastric volume are difficult to manage and are associated to poor quality-of-life. Stomach augmentation using a tissue-engineered stomach is a potential solution to restore adequate physiology and food reservoir. Aim of this study was to evaluate the decellularisation of whole rat stomach using a detergent-enzymatic protocol. METHODS Stomachs harvested from rats were decellularised through luminal and vascular cannulation using 24-h detergent-enzymatic treatment and completely characterized by appropriate staining, DNA and Extracellular matrix -component quantifications. RESULTS The detergent-enzymatic protocol allows a complete decellularisation of the gastric tissue, with a complete removal of the DNA with two cycles as confirmed by both quantifications and histological analysis. Extracellular matrix components, collagen, fibronectin, laminin and elastin, were optimally preserved by the treatment, while glycosaminoglycans were reduced. CONCLUSION Gastric tissue can be efficiently decellularised. Scaffolds retained original structure and important components that could enhance integration with other tissues for in vivo transplant. The use of naturally derived material could be potentially considered for the treatment of both congenital and acquired conditions.
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Affiliation(s)
- Elisa Zambaiti
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Federico Scottoni
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Eleonora Rizzi
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Simone Russo
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Koichi Deguchi
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Simon Eaton
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Alessandro F. Pellegata
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK
| | - Paolo De Coppi
- Stem Cell and Regenerative Medicine Section, DBC, UCL, Great Ormond Street Institute of Child Health, University College of London, Surgery Offices, 30 Guilford Street, London, WC1N 1EH UK ,Specialist Neonatal and Paediatric Surgery, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
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50
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Coronado RE, Somaraki-Cormier M, Natesan S, Christy RJ, Ong JL, Halff GA. Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison. Cell Transplant 2018; 26:1840-1854. [PMID: 29390876 PMCID: PMC5802637 DOI: 10.1177/0963689717742157] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized–solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.
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Affiliation(s)
| | | | - Shanmugasundaram Natesan
- 2 Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, JBSA-Fort Sam Houston, Sam Houston, TX, USA
| | - Robert J Christy
- 2 Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, JBSA-Fort Sam Houston, Sam Houston, TX, USA
| | - Joo L Ong
- 3 Biomedical Engineering San Antonio, University of Texas at San Antonio, San Antonio, TX, USA
| | - Glenn A Halff
- 4 Transplant Center San Antonio, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
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