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Zhou H, Liu H, Ezzelarab M, Schmelzer E, Wang Y, Gerlach J, Gridelli B, Cooper DKC. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature. Xenotransplantation 2015; 22:239-48. [PMID: 25950141 PMCID: PMC4519403 DOI: 10.1111/xen.12170] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2014] [Accepted: 04/18/2015] [Indexed: 12/11/2022]
Abstract
Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.
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Affiliation(s)
- Huidong Zhou
- Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
- Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Heng(1)yang, Hunan, China
| | - Hong Liu
- Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
- Department of General Surgery, First Hospital of Shanxi Medical University, ShanXi, China
| | - Mohamed Ezzelarab
- Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
| | - Eva Schmelzer
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
| | - Yi Wang
- Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Heng(1)yang, Hunan, China
| | - Jörg Gerlach
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
| | - Bruno Gridelli
- Mediterranean Institute for Transplantation and Advanced Specialized Therapies (ISMETT), Palermo, Italy
| | - David K. C. Cooper
- Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
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Loukopoulos I, Sfiniadakis I, Pillai A, Konstantoulakis M, Androulakis G, Bonatsos V, Zografos G, Papalois A. Mycophenolate Mofetil and Sirolimus in Hepatocyte Transplantation in an Experimental Model of Toxic Acute Liver Failure. J INVEST SURG 2014; 27:205-13. [DOI: 10.3109/08941939.2013.879967] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Lytras D, Papalois A, Tsaroucha AK, Papagoras D, Kyriazanos J, Lambropoulou M, Giannakou N, Galanos A, Simopoulos CE. Xenotransplantation of hepatocytes in rats with acute liver failure using sirolimus for immunosuppression. J Int Med Res 2010; 38:546-57. [PMID: 20515568 DOI: 10.1177/147323001003800217] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
This study aimed to evaluate the effect of sirolimus (SRL; rapamycin) as an immunosuppressant during xeno transplantation (XT) of rabbit hepatocytes into male Wistar rats with acute liver failure (ALF; n = 72). Isolated rabbit hepatocytes were transplanted intrasplenically into rats within 24 h of chemically induced ALF. Treatment groups received monotherapy with either cyclosporine (CsA) 20 mg/kg or SRL 0.20 mg/kg, or combination therapy with CsA 20 mg/kg + SRL 0.20 mg/kg for 14 days post-transplant. One control group with ALF received no treatment and a second group with ALF received only XT. Surviving rats were euthanized after 14 days, with concurrent blood sampling and organ retrieval for morphological evaluation. Survival rates at 14 days were: no XT/no treatment, 0%; XT alone, 29%; XT + CsA, 79%; XT + SRL, 33%; and XT + CsA + SRL, 33%. Liver morphology showed statistically superior liver regeneration for groups on SRL therapy. It is concluded that, in this hepatocyte XT model, SRL offered no survival advantage for ALF management so CsA still maintains a central role in attempts to develop alternative solutions for ALF.
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Affiliation(s)
- D Lytras
- Second Department of Surgery, Democritus University of Thrace, Alexandroupolis, Greece
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Abstract
Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative, or at least as a bridge, to orthotopic liver transplantation. The success of such a therapeutic approach remains limited by the quality of the transplanted cells. Cryopreservation remains the best option for long-term storage of hepatocytes, providing a permanent and sufficient cell supply. However, isolated adult hepatocytes are poorly resistant to such a process, with a significant alteration both at the morphological and functional levels. Hence, the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols, as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes. Intracellular ice formation or exposure to hyperosmotic solutions remains the main phenomenon of cryopreservation process, and its effects on cell quality and cell death induction will be discussed. The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing. Such strategies, such as vitrification, will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies.
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Papagoras D, Papalois A, Tsaroucha A, Lytras D, Kyriazanos J, Giannakou N, Laftsidis P, Simopoulos C. Beneficial effect of an antibody against interleukin-2 receptor (daclizumab) in an experimental model of hepatocyte xenotransplantation. World J Gastroenterol 2007; 13:1435-7. [PMID: 17457977 PMCID: PMC4146930 DOI: 10.3748/wjg.v13.i9.1435] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the use of Daclizumab (Dmab) as an immunosuppressive agent in an experimental model of hepatocyte xenotransplantation (XenoTx) in rats with fulminant hepatic failure (FHF).
METHODS: Two white male New Zealand rabbits were used as donors and 68 Wistar rats as recipients. FHF was induced by intravenous application of dimethylnitrosamine (DMNA). The isolated hepatocytes of the rabbits were xenotransplanted into the spleen of the rats 24 h after FHF induction. Group A (n = 13): no treatment; Group B (n = 14): FHF and XenoTx; Group C (n = 14): FHF and XenoTx and cyclosporin (CsA); Group D (n = 14): FHF and XenoTx and Dmab; Group E (n = 13): FHF and XenoTx and CsA and Dmab. The rats were followed for 15 d.
RESULTS: Statistical analysis showed better survival among groups D (92.86%) and E (76.92%) compared to group A (all rats died after 72 h), group B (28.57%) or group C (71.43%), although the differences were not statistically significant. Biochemical evaluation of the liver enzymes and histology confirmed satisfactory function and engraftment, respectively.
CONCLUSION: This experimental model has shown the safe, effective and beneficial use of Dmab in a xenotransplantation model of rabbit hepatocytes in rats.
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MESH Headings
- Acute Disease
- Animals
- Antibodies, Monoclonal/immunology
- Antibodies, Monoclonal/therapeutic use
- Antibodies, Monoclonal, Humanized
- Cell Transplantation/methods
- Daclizumab
- Disease Models, Animal
- Hepatocytes/transplantation
- Immunoglobulin G/immunology
- Immunoglobulin G/therapeutic use
- Immunosuppressive Agents/therapeutic use
- Liver Failure, Acute/pathology
- Liver Failure, Acute/surgery
- Male
- Rabbits
- Rats
- Rats, Wistar
- Receptors, Interleukin-2/antagonists & inhibitors
- Receptors, Interleukin-2/immunology
- T-Lymphocytes, Helper-Inducer/immunology
- T-Lymphocytes, Helper-Inducer/pathology
- Transplantation, Heterologous/immunology
- Transplantation, Heterologous/methods
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Affiliation(s)
- Dimitrios Papagoras
- 2nd Department of Surgery, Medical School, Democritus University of Thrace, Alexandroupolis, Greece
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Terry C, Dhawan A, Mitry RR, Hughes RD. Cryopreservation of isolated human hepatocytes for transplantation: State of the art. Cryobiology 2006; 53:149-59. [PMID: 16793034 DOI: 10.1016/j.cryobiol.2006.05.004] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2006] [Revised: 05/08/2006] [Accepted: 05/15/2006] [Indexed: 01/16/2023]
Abstract
Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.
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Affiliation(s)
- Claire Terry
- King's College London School of Medicine at King's College Hospital, Institute of Liver Studies, Bessemer Road, London SE5 9PJ, UK
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Tsiolis I, Papalois A, Loukopoulos I, Gravvanis A, Lykoudis E, Theodossopoulou E, Chairakakis A, Dimitroulopoulos D, Sfiniadakis I, Vassiliou I, Felekouras E, Dedeilias P, Kontogiorgi M, Papadimitriou L, Papadimitriou I. Experimental isolation and transplantation of hepatocytes with the use of antibody against interleukin-2 receptor (daclizumab) as immunosuppressive agent. Transplant Proc 2005; 37:1929-30. [PMID: 15919507 DOI: 10.1016/j.transproceed.2005.02.097] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
INTRODUCTION Daclizumab (Dmab) is a genetically engineered humanized IgG1 monoclonal antibody that binds to the alpha chain of the interleukin-2 receptor (Tac, CD25, p55) expressed on activated human T lymphocytes. Dmab has been used in a clinical protocol of islet transplantation with satisfactory results. The aim of the present study was to evaluate the use of an antibody against the interleukin-2 receptor (Dmab) as an immunosuppressive agent in an experimental model of hepatocyte allotransplantation (allo-Tx) in rats with fulminant hepatic failure (FHF). MATERIALS AND METHODS Six Wistar rats were used as donors and 48 Lewis rats as recipients: four groups of 12 animals each with induction of FHF and 24 hour later hepatocyte Tx--group A: no treatment; group B: cyclosporin (20 mg/kg days 0 to 5 and 10 mg/kg days 6 to 15); group C: Dmab (0.05 mg day of Tx and 0.05 mg day 7); and group D: Dmab and cyclosporine. Hepatocytes were transplanted intrasplenically. Animals were followed for 15 days. RESULTS Statistical analysis showed better survival among groups C (83%, MST = 13) and D (92%, MST = 14.25) compared to groups A (max 72, MST = 1.5) or B (50%, MST = 9). Survival in group D was better but not significantly than group C. Biochemical evaluation and histology confirmed satisfactory function and engraftment, respectively. CONCLUSION This experimental model showed the safe, effective use of Dmab.
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Affiliation(s)
- I Tsiolis
- Department of Surgery, E. Dynan Hospital, Athens, Greece
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Nagasaka S, Taniguchi S, Nakayama Y, Sakaguchi H, Nishizaki K, Naito H, Morioka H. In vivo study of the effects of cryopreservation on heart valve xenotransplantation. Cardiovasc Pathol 2005; 14:70-9. [PMID: 15780798 DOI: 10.1016/j.carpath.2005.01.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2003] [Revised: 09/01/2004] [Accepted: 01/06/2005] [Indexed: 11/26/2022] Open
Abstract
BACKGROUND Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.
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Affiliation(s)
- Shigeo Nagasaka
- Department of Thoracic and Cardiovascular Surgery, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan.
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Lloyd TDR, Orr S, Dennison AR. A Survey of Consumer Attitudes to the Supply and Use of Human Hepatocytes in the United Kingdom. Altern Lab Anim 2003; 31:483-8. [PMID: 15598175 DOI: 10.1177/026119290303100505] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Human hepatocytes are the model of choice for pharmacotoxicological studies, but their acquisition is often problematic due to ethical and logistical difficulties. The UK Human Tissue Bank is a not-for-profit organisation that acquires and processes human tissue, with a specialist interest in the isolation of human hepatocytes. A recent in-house survey of the processing of liver tissue over 1 year revealed that freshly isolated hepatocytes were underutilised due to mismatched consumer demand, despite the published need for them. We present the results of a telephone survey to investigate the reasons behind this paradox. This survey highlighted some problem areas, including “out of hours” availability of cells and personnel difficulties, but overall, demonstrated the value of such a service, with numerous researchers taking advantage of available good quality human hepatocytes. Although further work is required in optimising long-term storage protocols through cryopreservation, we have demonstrated that tissue handling of this type can be successful and beneficial to the pharmaceutical and biotechnology industries.
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Affiliation(s)
- Tom D R Lloyd
- Department of Surgery, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK
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Benoist S, Sarkis R, Chafaï N, Barbu V, Honiger J, Lakehal F, Becquemont L, Baudrimont M, Capeau J, Housset C, Nordlinger B. Survival and differentiation of porcine hepatocytes encapsulated by semiautomatic device and allotransplanted in large number without immunosuppression. J Hepatol 2001; 35:208-16. [PMID: 11580143 DOI: 10.1016/s0168-8278(01)00085-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
BACKGROUND/AIMS The aim of this study was to evaluate the survival and functions of porcine hepatocytes transplanted in large quantities in the peritoneal cavity of allogeneic animals following semiautomatic encapsulation. METHODS Isolated porcine hepatocytes and a polymer solution composed of AN69 were coextruded through a double lumen spinneret. Minitubes containing hepatocytes were transplanted in the peritoneal cavity of 12 pigs (4 x 10(9) cells/animal) in the absence of immunosuppressive therapy. Seven, 15, and 21 days after transplantation, minitubes was collected and processed for analyses. The morphology was examined under light and electron microscopy. Albumin synthesis was assessed by semi-quantitative reverse transcription-polymerase chain reaction. Cytochrome P450 3A (CYP3A) gene expression was analyzed by Western blot and by testosterone 6-beta-hydroxylation assay. RESULTS The device allowed to encapsulate 55 x 10(6) hepatocytes/min. Hepatocytes exhibited normal structural and ultrastructural features up to day 21. Albumin gene expression decreased progressively between days 0 and 21. The amount of CYP3A protein and 6-beta-hydroxylase activity were approximately 2-fold lower at days 7 and 15 than in freshly encapsulated hepatocytes, and further decreased thereafter. CONCLUSIONS The preservation of hepatocyte functions during 1-2 weeks is encouraging for potential short-term use of such bioartificial liver in future clinical application.
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Affiliation(s)
- S Benoist
- Research Unit 402, INSERM, Hospital Saint-Antoine, Paris, France
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Benoist S, Sarkis R, Barbu V, Honiger J, Baudrimont M, Lakehal F, Becquemont L, Delelo R, Housset C, Balladur P, Capeau J, Nordlinger B. Survival and functions of encapsulated porcine hepatocytes after allotransplantation or xenotransplantation without immunosuppression. Surgery 2001; 129:606-16. [PMID: 11331453 DOI: 10.1067/msy.2001.112961] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND This study evaluated the survival and functions of encapsulated porcine hepatocytes after intraperitoneal allotransplantation and xenotransplantation without immunosuppression. METHODS Isolated porcine hepatocytes were encapsulated in AN 69 polymer capsules (45.10(6)/capsule) and transplanted intraperitoneally in 12 rats and 12 pigs. Fifteen, 30, and 60 days after transplantation, capsules were removed and the viability and morphology of explanted hepatocytes were examined under light and electronic microscopy. The potential to produce albumin was assessed by evaluating the level of albumin messenger RNA, using semiquantitative reverse transcription-polymerase chain reaction. 6beta-Hydroxylase activity was measured by high-performance liquid chromatography. In addition, cytochrome P450 3A proteins were detected by Western blot only in allogeneic hepatocytes. RESULTS Similar results were observed after allotransplantation and xenotransplantation. Histologic studies showed that hepatocytes were well-preserved and arranged in cords for up to 30 days. The expression of porcine albumin gene was maintained up to 15 days. 6beta-Hydroxylase activity was 2.5-fold lower at day 15 than in freshly encapsulated hepatocytes, which were not transplanted. In allogeneic hepatocytes, the expression of CYP 3A protein was detected up to 60 days after transplantation. CONCLUSIONS Encapsulated porcine hepatocytes remain viable and functional for at least 15 days after allotransplantation and xenotransplantation without immunosuppression. The demonstration of maintained hepatic functions in transplanted porcine hepatocytes up to 15 days is a first step toward application in the treatment of acute liver failure.
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Affiliation(s)
- S Benoist
- Research Unit 402 of INSERM, Paris, France
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Auchincloss H. Literature update 1997, part 2. Xenotransplantation 1997. [DOI: 10.1111/j.1399-3089.1997.tb00192.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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