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Legallais C, Kim D, Mihaila SM, Mihajlovic M, Figliuzzi M, Bonandrini B, Salerno S, Yousef Yengej FA, Rookmaaker MB, Sanchez Romero N, Sainz-Arnal P, Pereira U, Pasqua M, Gerritsen KGF, Verhaar MC, Remuzzi A, Baptista PM, De Bartolo L, Masereeuw R, Stamatialis D. Bioengineering Organs for Blood Detoxification. Adv Healthc Mater 2018; 7:e1800430. [PMID: 30230709 DOI: 10.1002/adhm.201800430] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2018] [Revised: 08/23/2018] [Indexed: 12/11/2022]
Abstract
For patients with severe kidney or liver failure the best solution is currently organ transplantation. However, not all patients are eligible for transplantation and due to limited organ availability, most patients are currently treated with therapies using artificial kidney and artificial liver devices. These therapies, despite their relative success in preserving the patients' life, have important limitations since they can only replace part of the natural kidney or liver functions. As blood detoxification (and other functions) in these highly perfused organs is achieved by specialized cells, it seems relevant to review the approaches leading to bioengineered organs fulfilling most of the native organ functions. There, the culture of cells of specific phenotypes on adapted scaffolds that can be perfused takes place. In this review paper, first the functions of kidney and liver organs are briefly described. Then artificial kidney/liver devices, bioartificial kidney devices, and bioartificial liver devices are focused on, as well as biohybrid constructs obtained by decellularization and recellularization of animal organs. For all organs, a thorough overview of the literature is given and the perspectives for their application in the clinic are discussed.
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Affiliation(s)
- Cécile Legallais
- UMR CNRS 7338 Biomechanics & Bioengineering; Université de technologie de Compiègne; Sorbonne Universités; 60203 Compiègne France
| | - Dooli Kim
- (Bio)artificial organs; Department of Biomaterials Science and Technology; Faculty of Science and Technology; TechMed Institute; University of Twente; P.O. Box 217 7500 AE Enschede The Netherlands
| | - Sylvia M. Mihaila
- Division of Pharmacology; Utrecht Institute for Pharmaceutical Sciences; Utrecht University; Universiteitsweg 99 3584 CG Utrecht The Netherlands
- Department of Nephrology and Hypertension; University Medical Center Utrecht and Regenerative Medicine Utrecht; Utrecht University; Heidelberglaan 100 3584 CX Utrecht The Netherlands
| | - Milos Mihajlovic
- Division of Pharmacology; Utrecht Institute for Pharmaceutical Sciences; Utrecht University; Universiteitsweg 99 3584 CG Utrecht The Netherlands
| | - Marina Figliuzzi
- IRCCS-Istituto di Ricerche Farmacologiche Mario Negri; via Stezzano 87 24126 Bergamo Italy
| | - Barbara Bonandrini
- Department of Chemistry; Materials and Chemical Engineering “Giulio Natta”; Politecnico di Milano; Piazza Leonardo da Vinci 32 20133 Milan Italy
| | - Simona Salerno
- Institute on Membrane Technology; National Research Council of Italy; ITM-CNR; Via Pietro BUCCI, Cubo 17C - 87036 Rende Italy
| | - Fjodor A. Yousef Yengej
- Department of Nephrology and Hypertension; University Medical Center Utrecht and Regenerative Medicine Utrecht; Utrecht University; Heidelberglaan 100 3584 CX Utrecht The Netherlands
| | - Maarten B. Rookmaaker
- Department of Nephrology and Hypertension; University Medical Center Utrecht and Regenerative Medicine Utrecht; Utrecht University; Heidelberglaan 100 3584 CX Utrecht The Netherlands
| | | | - Pilar Sainz-Arnal
- Instituto de Investigación Sanitaria de Aragón (IIS Aragon); 50009 Zaragoza Spain
- Instituto Aragonés de Ciencias de la Salud (IACS); 50009 Zaragoza Spain
| | - Ulysse Pereira
- UMR CNRS 7338 Biomechanics & Bioengineering; Université de technologie de Compiègne; Sorbonne Universités; 60203 Compiègne France
| | - Mattia Pasqua
- UMR CNRS 7338 Biomechanics & Bioengineering; Université de technologie de Compiègne; Sorbonne Universités; 60203 Compiègne France
| | - Karin G. F. Gerritsen
- Department of Nephrology and Hypertension; University Medical Center Utrecht and Regenerative Medicine Utrecht; Utrecht University; Heidelberglaan 100 3584 CX Utrecht The Netherlands
| | - Marianne C. Verhaar
- Department of Nephrology and Hypertension; University Medical Center Utrecht and Regenerative Medicine Utrecht; Utrecht University; Heidelberglaan 100 3584 CX Utrecht The Netherlands
| | - Andrea Remuzzi
- IRCCS-Istituto di Ricerche Farmacologiche Mario Negri; via Stezzano 87 24126 Bergamo Italy
- Department of Management; Information and Production Engineering; University of Bergamo; viale Marconi 5 24044 Dalmine Italy
| | - Pedro M. Baptista
- Instituto de Investigación Sanitaria de Aragón (IIS Aragon); 50009 Zaragoza Spain
- Department of Management; Information and Production Engineering; University of Bergamo; viale Marconi 5 24044 Dalmine Italy
- Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas (CIBERehd); 28029 Barcelona Spain
- Fundación ARAID; 50009 Zaragoza Spain
- Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz; 28040 Madrid Spain. Department of Biomedical and Aerospace Engineering; Universidad Carlos III de Madrid; 28911 Madrid Spain
| | - Loredana De Bartolo
- Institute on Membrane Technology; National Research Council of Italy; ITM-CNR; Via Pietro BUCCI, Cubo 17C - 87036 Rende Italy
| | - Rosalinde Masereeuw
- Division of Pharmacology; Utrecht Institute for Pharmaceutical Sciences; Utrecht University; Universiteitsweg 99 3584 CG Utrecht The Netherlands
| | - Dimitrios Stamatialis
- (Bio)artificial organs; Department of Biomaterials Science and Technology; Faculty of Science and Technology; TechMed Institute; University of Twente; P.O. Box 217 7500 AE Enschede The Netherlands
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Sarika PR, James NR, Anilkumar PR, Raj DK, Kumary TV. Microgravity as a means to incorporate HepG2 aggregates in polysaccharide-protein hybrid scaffold. JOURNAL OF MATERIALS SCIENCE. MATERIALS IN MEDICINE 2016; 27:27. [PMID: 26704544 DOI: 10.1007/s10856-015-5638-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Accepted: 11/27/2015] [Indexed: 06/05/2023]
Abstract
Tissue culture under microgravity provides a venue which promotes cell-cell association while avoiding the detrimental effects of high shear stress. Hepatocytes cultured on carriers or entrapped within matrices under simulated microgravity conditions showed improved cell function and proliferation. In the present study, a new approach was adopted where a non-cell adherent scaffold was incorporated with hepatospheroids (HepG2) under microgravity. Gum arabic (GA) was cross-linked with gelatin (GA-Gel) and collagen (GA-Col) to prepare non-cell adherent scaffolds. Microgravity experiments with GA-Gel and GA-Col indicated that GA-Col is a better substrate compared to GA-Gel. Microgravity experiments of GA-Col scaffolds with HepG2 cells confirmed that the non-adherent surface with porous architecture can incorporate hepatocyte spheroids and maintain liver specific functions. Albumin and urea synthesis of hepatocytes was sustained up to 6 days under microgravity conditions in the presence of GA-Col scaffold. This new approach of using non-cell adherent matrix and microgravity environment for developing biological substitutes will be beneficial in tissue engineering, bioartificial liver devices and in vitro safety assessment of drugs.
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Affiliation(s)
- P R Sarika
- Department of Chemistry, Indian Institute of Space Science and Technology (IIST), Govt. of India, Valiamala, Thiruvananthapuram, 695 547, Kerala, India.
| | - Nirmala Rachel James
- Department of Chemistry, Indian Institute of Space Science and Technology (IIST), Govt. of India, Valiamala, Thiruvananthapuram, 695 547, Kerala, India.
| | - P R Anilkumar
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, 695 012, Kerala, India.
| | - Deepa K Raj
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, 695 012, Kerala, India.
| | - T V Kumary
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, 695 012, Kerala, India.
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Sarika PR, Sidhy Viha CV, Sajin Raj RG, Nirmala RJ, Anil Kumar PR. A non-adhesive hybrid scaffold from gelatin and gum Arabic as packed bed matrix for hepatocyte perfusion culture. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2014; 46:341-7. [PMID: 25491996 DOI: 10.1016/j.msec.2014.10.044] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 03/14/2014] [Revised: 09/27/2014] [Accepted: 10/21/2014] [Indexed: 10/24/2022]
Abstract
Development of liver support systems has become one of the most investigated areas for the last 50 years because of the shortage of donor organs for orthotopic liver transplantations. Bioartificial liver (BAL) device is one of the alternatives for liver failure which provides a curing method and support patients to recover from certain liver failure diseases. The biological compartment of BAL is called the bioreactor where functionally active hepatocytes are maintained to support the liver specific functions. We have developed a packed bed bioreactor with a cytocompatible, polysaccharide-protein hybrid scaffold. The scaffold prepared from gelatin and gum Arabic acts as a packed bed matrix for hepatocyte culture. Quantitative evaluation of the hepatocytes cultured using packed bed bioreactor demonstrated that cells maintained liver specific functions like albumin and urea synthesis for seven days. These results indicated that the system can be scaled up to form the biological component of a bioartificial liver.
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Affiliation(s)
- P R Sarika
- Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547, India
| | - C V Sidhy Viha
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695 012, India
| | - R G Sajin Raj
- Device Testing Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695 012, India
| | - Rachel James Nirmala
- Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547, India
| | - P R Anil Kumar
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695 012, India.
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Yadav N, Kanjirakkuzhiyil S, Ramakrishnan M, Das TK, Mukhopadhyay A. Factor VIII can be synthesized in hemophilia A mice liver by bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells. Stem Cells Dev 2011; 21:110-20. [PMID: 21480781 DOI: 10.1089/scd.2010.0569] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Hemophilia A (HA) is caused by mutation in factor VIII (FVIII) gene in humans; it leads to inadequate synthesis of active protein. Liver is the primary site of FVIII synthesis; however, the specific cell types responsible for its synthesis remain controversial. We propose that the severity of the bleeding disorder could be ameliorated by partial replacement of mutated liver cells by healthy cells in HA mice. The aim of this investigation was to study the cellular origin of FVIII by examining bone marrow cell therapy for treatment of HA in mice. Recipient liver was perturbed with either acetaminophen or monocrotaline to facilitate the engraftment and differentiation of lineage-depleted (Lin(-)) enhanced green fluorescent protein-expressing bone marrow cells. Immunohistochemical analysis of liver tissue was conducted to identify the donor-derived cells that expressed FVIII. This identification was confirmed by transmission electron microscopy and quantitative gene expression analysis. The phenotypic correction in HA mice was determined by tail-clip challenge and FVIII level in plasma by Chromogenix and activated partial thromboplastin time assays. Immunohistochemical analysis showed that von Willebrand factor and cytokeratin-18-expressing endothelial cells and hepatocytes, respectively, were obtained from BM-derived cells. Both cell types expressed FVIII light chain mRNA and protein, which was further confirmed by transmission electron microscopy. The transplanted HA mice showed FVIII activity in plasma (P<0.01) and survived tail-clip challenge (P<0.001). Thus, we conclude that BM-derived hepatocytes and endothelial cells can synthesize FVIII in liver and correct bleeding phenotype in HA mice.
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Affiliation(s)
- Neelam Yadav
- Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
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Elliott NT, Yuan F. A review of three-dimensional in vitro tissue models for drug discovery and transport studies. J Pharm Sci 2010; 100:59-74. [PMID: 20533556 DOI: 10.1002/jps.22257] [Citation(s) in RCA: 326] [Impact Index Per Article: 21.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2009] [Accepted: 05/04/2010] [Indexed: 12/12/2022]
Abstract
The use of animal models in drug discovery studies presents issues with feasibility and ethical concerns. To address these limitations, in vitro tissue models have been developed to provide a means for systematic, repetitive, and quantitative investigation of drugs. By eliminating or reducing the need for animal subjects, these models can serve as platforms for more tightly controlled, high-throughput screening of drugs and for pharmacokinetic and pharmacodynamic analyses of drugs. The focus of this review is three-dimensional (3D) tissue models that can capture cell-cell and cell-matrix interactions. Compared to the 2D culture of cell monolayers, 3D models more closely mimic native tissues since the cellular microenvironment established in the 3D models often plays a significant role in disease progression and cellular responses to drugs. A growing body of research has been published in the literature, which highlights the benefits of the 3D in vitro models of various tissues. This review provides an overview of some successful 3D in vitro models that have been developed to mimic liver, breast, cardiac, muscle, bone, and corneal tissues as well as malignant tissues in solid tumors.
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Affiliation(s)
- Nelita T Elliott
- Department of Biomedical Engineering, Duke University, 136 Hudson Hall, PO Box 90281, Durham, North Carolina 27708, USA
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Abstract
A variety of bioartificial liver support systems were developed to replace some of the liver's function in case of liver failure. Those systems, in contrast to purely artificial systems, incorporate metabolically active cells to contribute synthetic and regulatory functions as well as detoxification. The selection of the ideal cell source and the design of more sophisticated bioreactors are the main issues in this field of research. Several systems were already introduced into clinical studies to prove their safety. This review briefly introduces a cross-section of experimental and clinically applied systems and tries to give an overview on the problems and limitations of bioartificial liver support.
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Affiliation(s)
- Gesine Pless
- Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany
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The therapeutic effect of bone marrow-derived liver cells in the phenotypic correction of murine hemophilia A. Blood 2009; 114:4552-61. [PMID: 19752394 DOI: 10.1182/blood-2009-02-202788] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The transdifferentiation of bone marrow cells (BMCs) into hepatocytes has created enormous interest in applying this process to the development of cellular medicine for degenerative and genetic diseases. Because the liver is the primary site of factor VIII (FVIII) synthesis, we hypothesized that the partial replacement of mutated liver cells by healthy cells in hemophilia A mice could manage the severity of the bleeding disorder. We perturbed the host liver with acetaminophen to facilitate the engraftment and hepatic differentiation of lineage-depleted enhanced green fluorescent protein-expressing BMCs. Immunohistochemistry experiments with the liver tissue showed that the donor-derived cells expressed the markers of both hepatocytes (albumin and cytokeratin-18) and endothelial cells (von Willebrand factor). The results of fluorescent in situ hybridization and immunocytochemistry experiments suggested that differentiation was direct in this model. The BMC-recipient mice expressed FVIII protein and survived in a tail clip challenge experiment. Furthermore, a coagulation assay confirmed that the plasma FVIII activity was maintained at 20.4% (+/- 3.6%) of normal pooled plasma activity for more than a year without forming its inhibitor. Overall, this report demonstrated that BMCs rescued the bleeding phenotype in hemophilia A mice, suggesting a potential therapy for this and other related disorders.
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Li JG, Chen YK, Wang YM. Splicing of SV40T gene exons and construction of a retroviral vector pLLTSN. Shijie Huaren Xiaohua Zazhi 2004; 12:1104-1107. [DOI: 10.11569/wcjd.v12.i5.1104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct an immortalization vector for hepatocytes immortalization, and two exons of simian virus 40 large T antigen gene (SV40T) were spliced and a retroviral vector pLLTSN without intron was constructed.
METHODS: The two exons of SV40T were amplified respectively by high fidelity polymerase chain reaction (PCR) by using the plasmid pUC19-SV40T as the template. Then SV40T gene was spliced by overlapping extension (SOE), and cloned into theEcoRⅠand BamHⅠsites of the retroviral vector pLXSN. The positive recombinant clones were screened and identified by PCR by using colonies directly as templates, and by restriction endonuclease digestion analysis, and DNA sequence analysis.
RESULTS: The 2.1 kb SV40T gene was spliced. Among the ten colonies randomly screened, four were proved positive, and one of them was verified by plasmid DNA sequencing.
CONCLUSION: The retroviral vector pLLTSN containing SV40T without intron is successfully constructed.
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Di Campli C, Gasbarrini G, Gasbarrini A. Review article: a medicine based on cell transplantation -- is there a future for treating liver diseases? Aliment Pharmacol Ther 2003; 18:473-80. [PMID: 12950419 DOI: 10.1046/j.1365-2036.2003.01692.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Cell-based therapies, comprising isolated hepatocyte transplantation, ex vivo gene therapy and bioartificial liver devices, may actually design a new scenario in the treatment of patients with liver failure. Recent advances in the liver repopulation field and the considerable excitement surrounding the use of haematopoietic stem cells for a 'regenerative medicine', allow us to see under a new light the alternative cell sources actually under investigation. In particular, the major drawbacks and the most important advantages of xenogenic primary cells, tumour-derived cell lines, immortalised hepatocytes and stem cells in the wide range of experimental and clinical applications are presented. Even if up to now none of them represent a 'gold standard' in the clinical practice, the intersecting advances in the development of mechanical components of artificial devices and in the liver biology and bioengineering will open tantalising possibilities to treat patients with liver failure, by tailoring the therapeutic choice on the basis of the aetiology and the stage of liver disease.
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Affiliation(s)
- C Di Campli
- Department of Internal Medicine, Catholic University of Rome, Rome, Italy
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Guo R, Zou P, Fan HH, Gao F, Shang QX, Cao YL, Lu HZ. Repression of allo-cell transplant rejection through CIITA ribonuclease P + hepatocyte. World J Gastroenterol 2003; 9:1077-81. [PMID: 12717860 PMCID: PMC4611376 DOI: 10.3748/wjg.v9.i5.1077] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Allo-cell transplant rejection and autoimmune responses were associated with the presence of class II major histocompatibility complex (MHC II) molecules on cells. This paper studied the effect of Ribonuclease P (RNase P) against CIITA, which was a major regulator of MHCII molecules, on repressing the expression of MHCII molecules on hepatocyte.
METHODS: M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coli. It were constructed that M1-RNA with guide sequences (GS) recognizing the 452, 3408 site of CIITA by PCR from pTK117 plasmid, then were cloned into the EcoR I/Bgl II or EcoR I/Sal I site of vector psNAV (psNAV-M1-452-GS, psNAV-M1-3408-GS) respectively. The target mould plate (3176-3560) of CIITA was obtained from Raji cell by RT-PCR, and then inserted into the Xho I/EcoR I of pGEM-7zf(+) plasmid (pGEM-3176). These recombinant plasmids were screened out by sequence analysis. psNAV-M1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vitro. It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS. Stable transfectants of hepatocyte cell line with psNAV-M1-3408-GS were tested for expression of class II MHC through FCM, for mRNA abundance of MHCII, Ii and CIITA by RT-PCR, for the level of IL-2 mRNA on T cell by mixed lymphocyte reaction.
RESULTS: When induced with recombinant human interferon-gamma (IFN-γ), the expression of HLA-DR, -DP, -DQ on psNAV-M1-3408-GS+ hepatocyte was reduced 83.27%, 88.93%, 58.82% respectively, the mRNA contents of CIITA, HLA-DR, -DP, -DQ and Ii decreased significantly. While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS+ hepatocyte.
CONCLUSION: The Ribonuclease P against CIITA-M1-3408-GS could effectively induce antigen-specific tolerance through cleaving CIITA. These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and autoimmune diseases.
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Affiliation(s)
- Rong Guo
- Institute of Hematology, the Union Hospital, Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
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Abstract
AIM: To study the viscoelastic properties of human hepatocytes and hepatocellular carcinoma (HCC) cells under cytoskeletal perturbation, and to further to study the viscoelastic properties and the adhesive properties of mouse hepatoma cells (HTC) in different cell cycle.
METHODS: Micropipette aspiration technique was adopted to measure viscoelastic coefficients and adhesion force to collagen coated surface of the cells. Three kinds of cytoskeleton perturbing agents, colchicines (Col), cytochalasin D (CD) and vinblastine (VBL), were used to treat HCC cells and hepatocytes and the effects of these treatment on cell viscoelastic coefficients were investigated. The experimental results were analyzed with a three-element standard linear solid. Further, the viscoelastic properties of HTC cells and the adhesion force of different cycle HTC cells were also investigated. The synchronous G1 and S phase cells were achieved through thymine-2-desoryriboside and colchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively.
RESULTS: The elastic coefficients, but not viscous coefficient of HCC cells (K1 = 103.6 ± 12.6 N·m-2, K2 = 42.5 ± 10.4 N·m-2, μ = 4.5 ± 1.9 Pa·s), were significantly higher than the corresponding value for hepatocytes (K1 = 87.5 ± 12.1 N·m-2, K2 = 33.3 ± 10.3 N·m-2, μ = 5.9 ± 3.0 Pa·s, P < 0.01). Upon treatment with CD, the viscoelastic coefficients of both hepatocytes and HCC cells decreased consistently, with magnitudes for the decrease in elastic coefficients of HCC cells (K1: 68.7 N·m-2 to 81.7 N·m-2, 66.3% to 78.9%; K2: 34.5 N·m-2 to 37.1 N·m-2, 81.2% to 87.3%, P < 0.001) larger than those for normal hepatocytes (K1: 42.6 N·m-2 to 49.8 N·m-2, 48.7% to 56.9%; K2: 17.2 N·m-2 to 20.4 N·m-2, 51.7% to 61.3%, P < 0.001). There was a little decrease in the viscous coefficient of HCC cells (2.0 to 3.4 Pa•s, 44.4 to 75.6%, P < 0.001) than that for hepatocytes (3.0 to 3.9 Pa•s, 50.8 to 66.1% P < 0.001). Upon treatment with Col and VBL, the elastic coefficients of hepatocytes generally increased or tended to increase while those of HCC cells decreased. HTC cells with 72.1% of G1 phase and 98.9% of S phase were achieved and high K1, K2 value and low μ value were the general characteristics of HTC cells. G1 phase cells had higher K1 value and lower μ value than S phase cells had, and G1 phase HTC cells had stronger adhesive forces [(275.9 ± 232.8) × 10-10 N] than S phase cells [(161.2 ± 120.4) × 10-10 N, P < 0.001).
CONCLUSION: The difference in both the pattern and the magnitude of the effect of cytoskeletal perturbing agent on the viscoelastic properties between HCC cells and hepatocytes may reflect differences in the state of the cytoskeleton structure and function and in the sensitivity to perturbing agent treatment between these two types of cells. Change in the viscoelastic properties of cancer cells may affect significantly tumor cell invasion and metastasis as well as interactions between tumor cells and their micro-mechanical environments.
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Affiliation(s)
- Gang Zhang
- Department of Pathophysiology, The Third Military Medical University, Chongqing 400038, China.
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Hoekstra R, Chamuleau RAFM. Recent developments on human cell lines for the bioartificial liver. Int J Artif Organs 2002; 25:182-91. [PMID: 11999190 DOI: 10.1177/039139880202500304] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Most bioartificial liver (BAL) devices contain porcine primary hepatocytes as their biological component. However, alternatives are needed due to xenotransplantation associated risks. Human liver cell lines have excellent growth characteristics and are therefore candidates for application in BAL devices. Tumour-derived cell lines HepG2 and C3A express a variety of liver functions, but some specific liver functions, like ammonia detoxification and ureagenesis are insufficient. Immortalised human hepatocytes might offer better prospects. The balance between immortalisation and transformation with dedifferentiation of cells seems controllable by conditional immortalisation and/or the use of telomerase as immortalising agent. Another promising approach will be the use of embryonic or adult human stem cells. Rodent stem cells have been directed to hepatic differentiation in vitro, which might be applicable to human stem cells. However, both functionality and safety of immortalised human liver cell lines and differentiated stem cells should be improved before successful use in BAL devices becomes reality.
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Affiliation(s)
- R Hoekstra
- Department of Experimental Hepatology, Academic Medical Center, University of Amsterdam, The Netherlands.
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Hou GQ, Liang XL, Chen R, Tang LW, Wang Y, Xu PY, Zhang YR, Ou CH. Copper transportion of WD protein in hepatocytes from Wilson disease patients in vitro. World J Gastroenterol 2001; 7:846-51. [PMID: 11854914 PMCID: PMC4695607 DOI: 10.3748/wjg.v7.i6.846] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/12/2001] [Revised: 09/19/2001] [Accepted: 09/28/2001] [Indexed: 02/06/2023] Open
Abstract
AIM To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD). METHODS WD copper transporting properties in some organelles of the cultured hepatocytes were studied from WD patients and normal controls.These cultured hepatocytes were incubated in the media of copper 15 mg x L(-1) only, copper 15 mg x L(-1) with vincristine (agonist of P-type ATPase) 0.5mg x L(-1), or copper 15 mg x L(-1) with vanadate (antagonist of P-type ATPase) 18.39 mg x L(-1) separately. Microsome (endoplasmic reticulum and Golgi apparatus), lysosome, mitochondria, and cytosol were isolated by differential centrifugation. Copper contents in these organelles were measured with atomic absorption spectrophotometer, and the influence in copper transportion of these organelles by vanadate and vincristine were comparatively analyzed between WD patients and controls. WD copper transporting P-type ATPase was detected by SDS-PAGE in conjunction with Western blot in liver samples of WD patients and controls. RESULTS The specific WD proteins (M(r)155,000 lanes) were expressed in human hepatocytes, including the control and WD patients. After incubation with medium containing copper for 2 h or 24 h, the microsome copper concentration in WD patients was obviously lower than that of controls, and the addition of vanadate or vincristine would change the copper transporting of microsomes obviously. When incubated with vincristine, levels of copper in microsome were significantly increased, while incubated with vanadate, the copper concentrations in microsome were obviously decreased. The results indicated that there were WD proteins, the copper transportion P-type ATPase in the microsome of hepatocytes. WD patients possessed abnormal copper transporting function of WD protein in the microsome, and the agonist might correct the defect of copper transportion by promoting the activity of copper transportion P-type ATPase. CONCLUSION Copper transportion P-type ATPase plays an important role in hepatocytic copper metabolism. Dysfunction of hepatocytic WD protein copper transportion might be one of the most important factors for WD.
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Affiliation(s)
- G Q Hou
- Department of Neurology, Guangzhou First Municipal People's Hospital, Guangzhou Medical College, Guangdong Province, China.
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Cao XY, Liu J, Lian ZR, Clayton M, Hu JL, Zhu MH, Fan DM, Feitelson M. Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice. World J Gastroenterol 2001; 7:575-8. [PMID: 11819834 PMCID: PMC4688678 DOI: 10.3748/wjg.v7.i4.575] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Affiliation(s)
- X Y Cao
- Institute of Digestive Diseases, Xijing Hospital, Xi'an 710033, Shaanxi Province, China.
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Wei HS, Li DG, Lu HM, Zhan YT, Wang ZR, Huang X, Zhang J, Cheng JL, Xu QF. Effects of AT1 receptor antagonist, losartan, on rat hepatic fibrosis induced by CCl 4. World J Gastroenterol 2000; 6:540-545. [PMID: 11819643 PMCID: PMC4723553 DOI: 10.3748/wjg.v6.i4.540] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate effect o f losartan, an AT1 receptor antagonist, on hepatic fibrosis induced by CCl4; and to determine whether or not AT1 receptors are expressed on hepatic stellate cells.
METHODS AND RESULTS: Fifty male Sprague-Dawley rats, weighing (180 ± 20) g, were randomized into five groups (control group, model group, and three los artan treated groups), in which all rats were given the subcutaneous injection o f 40% CCl4 (every 3 days for 6 weeks) except for rats of control group. Rats of losartan-treated groups were treated with losartan (20 mg/kg, 10 mg/kg, 5 mg/kg, daily gavage). After 6 weeks liver tissue and serum samples of all rats were examined. Serum hyaluronic acid (HA), procollagen type III (PC III) were detected by radioimmunoassays. van Giesion collagen staining was used to evaluate the extracellular matrix of rats with liver fibrosis. The expression of AT1 receptors, transforming growth factor-beta (TGF-β), and alpha-smooth muscle actin (α-SMA) in liver tissue were determined by immunohistochemical techniques. Compared with model group, serum ALT and AST of losartan-treated groups were significantly reduced (t = 4. 20, P < 0.01 and t = 4.57, P < 0.01). Serum HA and PC III also had significant differences (t = 3.53, P < 0.01 and t = 2.20, P < 0.05). The degree of fibrosis was improved by losartan and correlated with the expressions of AT1 receptors, TGF-β, and α-SMA in liver tissue.
CONCLUSION: AT1 receptor antagonist, losartan, could limit the progression of the hepatic fibrosis induced by CCl4. The mechanism may be relat ed to the decrease in the expression of AT1 receptors and TGF-β, a meliorating the injury of hepatocytes; activation of local renin-angiotensin system might relate to hepatic fibrosis; and during progression of fibrosis, activated hepatic stellate cells might express AT1 receptors.
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Wang YJ, Li MD, Wang YM, Chen GZ, Lu GD, Tan ZX. Effect of extracorporeal bioartificial liver support system on fulminant hepatic failure rabbits. World J Gastroenterol 2000; 6:252-254. [PMID: 11819568 PMCID: PMC4723496 DOI: 10.3748/wjg.v6.i2.252] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the possibility of using cultured human hepatocytes as a bridge between bioartificial liver and liver transplantation.
METHODS: In this experiment, the efficacy of extracorporeal bio artificial liver support system (EBLSS) consisting of spheriodal human liver cells and cultured hepatocytes supernatant was assessed in vivo using galactosamine induced rabbit model of fulminant hepatic failure.
RESULTS: There was no difference of survival between the two groups of rabbits, but in the supported rabbits serum alanine aminotransferase, total bilirubin and creatinine were significantly lower and hepatocyte necrosis was markedly milder than those in control animals. In addition, a good viability of human liver cells was noted after the experiment.
CONCLUSION: EBLSS plays a biologic role in maintaining and compensating the function of the liver.
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