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Isolation, culture and transplantation of pig fetal cardiomyocytes
3rd Intl Congress of Cell Transplant Society • ABSTRACTS
3.07
HIGIILY POROUS POLYMER MATRICES AS A THREEDIMENSIONAL CULTURE SYSTEM FOR HEPATOCYTES: INITIAL RESULTS
3.08
In this study, reendothelialization of expanded polytetrafluorcetbylene (ePTFE) grafts was studied. The ePTFE was precoated with human serum or collagen I. We have chosen serum for e ~ e n t u i and clinical proposes because of its awilability and safety. As comparative matrix we used collagenI, since this is known to mediate rapid and firm adhesion. Cultured adult human great saphenous vein endothelial cells (HSVECs) were seeded to conlluency on the precoated ePTFE. Reendothelialization was studied by exposing HSVECs to a cell-flee, precoated area (4xl0mm) for 3, 10 or 15 days. We also studied the deposition of basement membrane components (laminin and collagen IV) and the development of cell-cell contacts with a marker for a gap junction protein (connexin 43), 6h, 12h and 24h after confluent seeding of HSVECs on the precoated ePTFE. Initiation of migration was seen after 3 days, with the collagen I-couted elrI"FE endothelialized to a larger extent when compared to the seram--precoated graft. Complete reendothelialiTa~on of the collagen I-precented area was seen after 10 days. On the senunpreceated cPTFE, the reendotheliuiization-processwas shown to be somewhat slower as the HSVECs were confluent on day 15. The development of laminin, collagen IV and connexin 43 showed a similar time-course on both types of preenated ePTFE and the immunostaining of these proteins were detected as early as 6h postseeding with a staining becoming more intense up to 24h. Taken together, these findings suggest HSVECs seeded on serum- or collagen I-preconted ePTFE to be able to reendethefialize denudations possibly occurring at implantation and/or after exposure to flow. The data also indicate HSVECs to regenera~ a basement membrane and intercellular contacts. This may be of clinical importance for EC retention and the functional properties of endothelialized synthetic grafts implanted in matt
Cell tran.splantation as a ~erapyfor end-stage liver disease is currenuy unoer investigation oue to the donor ~ t y for organ transplantation. We have previously demonstrated that highly porous, three-dimensional matrices are suitable vehicles for liepatocyte transplantation. In this study, we have investigated the suitability of these matrices as a novel system for extended hepatocyte culture. . Polymer matrices (95% porous, pore size250-400 nun, overall oimensiou~ ixlx0.3cm) were fabricated from Ix~ly(L-lactic acid) (PLA) utitiziug a particulate ieachin~ technique. We investigated five different sl)onge coating condiuons: no coathlg, pluromc surfacrant and polyvmyl a l ~ h o l coating, collagen monolayer coating, and collagen gel coating. Conventional tissue culture flasks servedas consols. Hepatocytes were harvested from male Lewis rots using a s t e n c i l couagenase pcrmsiou method, and seeded onto matrices (5x 10" cellshuatrix).Tlie viability of cultured cells was assessed by, trypan blue exelusiou, and general metabolic activity by reduction ol 3-1.4,.~5-dimethylthiazol-zylJ-2,5-diphenyl tetrazolium bromide (M IT). Liver-specific pbenotype was analyzed by quantitating albumin secretion. Cultured lie_patocytes were observed by scanning electron microscopy (SEM) of fixed cell seeded matrices. In the tissue culture control all l)epatocytes detached and were lost within 7 days of seeding. In contrast, bepatocytes cultured on polymer matrices with all coating coeditious were malnmilled in culture for the 14 days of the experiment. SEM observation of these cultured revealed hepalocytes aallerent to the polymer matrices and cell-to-ceil adhesions. Allcolls cultured in polymer matrices mainmined metabolic activity, although liepatocytes cultured within collagen gels on polymer ntatrices exhibited the greatest metabolic activity (darkest staining with M'IT assay). Cells cultured on threedimensiolml polymer matrices exhibited liver-spocific gene expression over the duration of tile experhnent, as evidenced by albumin secretion, allhough secretion rates decreased with time ill culture. llepatocytes can be successfully cultured for extended times within porous timee-dintensional p21ymer matrices. Cell number, metabolic activity, and liver-sl~,ctfic function is maintained in this culture system, and can be regulated by the surface coating of the polymer matrices.
3.09
ISOLATION, CULTURE AND TRANSPLANTATION OF PIG FETAL CARDIOMYOCYTES
Zawadzka A, Ratliff J, Beaulieu L, Field L*, Dinsmore J, Boston I n d i a n a p o l i s * U S A An inability of myocardium to regenerate itself has prompted studies of cell transplantation into heart muscle to repair injury. Other researches have shown that fetal mouse cardiomyocytes grafted into synegeneic hosts form stable grafts that integrate into donor heart tissue and form cell-cell connections appropriate for adult cardiomyocytes. These studies showed the feasibility of such cardiac grafts and demonstxated the ability of transplanted cardiomyocytes to contribute to myocardial function. We have extended these studies to a xenograft model where fetal pig cardiomyocytes have been transplanted to recipient mice. We have used early gestation day pig fetuses to isolate pure populations of cardiomyocytes as identified by immunohistochemical staining of sarcomeric proteins (cardiac uoponin and myosin heavy chain). Isolated fetal pig cardiomyocytes show a limited potential for proliferation postisolation, consistent with what others have shown for fetal cardiomyocytes from other species. Culture conditions for growth, maintenance and contractility of cardiomyocytes were established. Cultured fetal pig cardiomyocytes masked with F(ab')2 fragments of anti pig class I antibody were injected directly into mouse myocardium. Implanted masked cardiomyecytes formed stable grafts over 30 day period, as evidenced by PRE (pig repeating element) in situ hybridization and PCR-PRE techniques.
HUMAN ENDOTHELIAL CELLS SHOW DIPI:P.:RENTIATED FUNCTIONS AFTER IN VITRO SEEDING ON eFIYE Jansson K, Bengtsson L, Hacgerstrand A; Stockholm SWEDEN
3.10
IMPROVED SURVIVAL IN ANHEPATIC RAT BY TRANSPLANTED HEPATOCYTES Lorenzo M, Angrisunl L, Di Prisco B, Cuomo 1l, Tesanro B; Naples Italy Aim of this study is to evaluate if hepatocytes U'ansplanted into the spleen may improve the survival in anhepatic rat, with any kind of concomitant pharmacologic support. A total hepntectomy was performed with an original method in which the liver was replaced by an heterologous
vasc~ar prosthe~ that joins the s~rahepaac cave. the subhepadc cuva and the portal vein. For all experiments male Sprague-Dawley rats (250-350 gr) were used. Hepatocytes (4x107) were transplanted by direct injection into the splenic parenchyma. Rats were allocated in the following groups: Group A (n=I0) that underwent a total hepatectomy only (control group); Group B (n=10), Group C (n=10) and Group D (n=10), that underwent hepatocyte transplantation respectively I, 2, and 9 months before of total hepatectomy. Statistical analysis was performed by means of Fisher's exact test and Student t-test. Survival of anlaepntic rats was significantly improved only in Group D rats, respect to control group(75+18 Vs 32+15; p<0.001). This fact indicate the need for hepatocytes to proliferate and colonize the engrafted organ. This result can be considered an useful tool to study the possibility to use hepatocyte trannsplantation as a bridge for orthotopic liver transplantation.
23
3.07
HIGIILY POROUS POLYMER MATRICES AS A THREEDIMENSIONAL CULTURE SYSTEM FOR HEPATOCYTES: INITIAL RESULTS
3.08
In this study, reendothelialization of expanded polytetrafluorcetbylene (ePTFE) grafts was studied. The ePTFE was precoated with human serum or collagen I. We have chosen serum for e ~ e n t u i and clinical proposes because of its awilability and safety. As comparative matrix we used collagenI, since this is known to mediate rapid and firm adhesion. Cultured adult human great saphenous vein endothelial cells (HSVECs) were seeded to conlluency on the precoated ePTFE. Reendothelialization was studied by exposing HSVECs to a cell-flee, precoated area (4xl0mm) for 3, 10 or 15 days. We also studied the deposition of basement membrane components (laminin and collagen IV) and the development of cell-cell contacts with a marker for a gap junction protein (connexin 43), 6h, 12h and 24h after confluent seeding of HSVECs on the precoated ePTFE. Initiation of migration was seen after 3 days, with the collagen I-couted elrI"FE endothelialized to a larger extent when compared to the seram--precoated graft. Complete reendothelialiTa~on of the collagen I-precented area was seen after 10 days. On the senunpreceated cPTFE, the reendotheliuiization-processwas shown to be somewhat slower as the HSVECs were confluent on day 15. The development of laminin, collagen IV and connexin 43 showed a similar time-course on both types of preenated ePTFE and the immunostaining of these proteins were detected as early as 6h postseeding with a staining becoming more intense up to 24h. Taken together, these findings suggest HSVECs seeded on serum- or collagen I-preconted ePTFE to be able to reendethefialize denudations possibly occurring at implantation and/or after exposure to flow. The data also indicate HSVECs to regenera~ a basement membrane and intercellular contacts. This may be of clinical importance for EC retention and the functional properties of endothelialized synthetic grafts implanted in matt
Cell tran.splantation as a ~erapyfor end-stage liver disease is currenuy unoer investigation oue to the donor ~ t y for organ transplantation. We have previously demonstrated that highly porous, three-dimensional matrices are suitable vehicles for liepatocyte transplantation. In this study, we have investigated the suitability of these matrices as a novel system for extended hepatocyte culture. . Polymer matrices (95% porous, pore size250-400 nun, overall oimensiou~ ixlx0.3cm) were fabricated from Ix~ly(L-lactic acid) (PLA) utitiziug a particulate ieachin~ technique. We investigated five different sl)onge coating condiuons: no coathlg, pluromc surfacrant and polyvmyl a l ~ h o l coating, collagen monolayer coating, and collagen gel coating. Conventional tissue culture flasks servedas consols. Hepatocytes were harvested from male Lewis rots using a s t e n c i l couagenase pcrmsiou method, and seeded onto matrices (5x 10" cellshuatrix).Tlie viability of cultured cells was assessed by, trypan blue exelusiou, and general metabolic activity by reduction ol 3-1.4,.~5-dimethylthiazol-zylJ-2,5-diphenyl tetrazolium bromide (M IT). Liver-specific pbenotype was analyzed by quantitating albumin secretion. Cultured lie_patocytes were observed by scanning electron microscopy (SEM) of fixed cell seeded matrices. In the tissue culture control all l)epatocytes detached and were lost within 7 days of seeding. In contrast, bepatocytes cultured on polymer matrices with all coating coeditious were malnmilled in culture for the 14 days of the experiment. SEM observation of these cultured revealed hepalocytes aallerent to the polymer matrices and cell-to-ceil adhesions. Allcolls cultured in polymer matrices mainmined metabolic activity, although liepatocytes cultured within collagen gels on polymer ntatrices exhibited the greatest metabolic activity (darkest staining with M'IT assay). Cells cultured on threedimensiolml polymer matrices exhibited liver-spocific gene expression over the duration of tile experhnent, as evidenced by albumin secretion, allhough secretion rates decreased with time ill culture. llepatocytes can be successfully cultured for extended times within porous timee-dintensional p21ymer matrices. Cell number, metabolic activity, and liver-sl~,ctfic function is maintained in this culture system, and can be regulated by the surface coating of the polymer matrices.
3.09
ISOLATION, CULTURE AND TRANSPLANTATION OF PIG FETAL CARDIOMYOCYTES
Zawadzka A, Ratliff J, Beaulieu L, Field L*, Dinsmore J, Boston I n d i a n a p o l i s * U S A An inability of myocardium to regenerate itself has prompted studies of cell transplantation into heart muscle to repair injury. Other researches have shown that fetal mouse cardiomyocytes grafted into synegeneic hosts form stable grafts that integrate into donor heart tissue and form cell-cell connections appropriate for adult cardiomyocytes. These studies showed the feasibility of such cardiac grafts and demonstxated the ability of transplanted cardiomyocytes to contribute to myocardial function. We have extended these studies to a xenograft model where fetal pig cardiomyocytes have been transplanted to recipient mice. We have used early gestation day pig fetuses to isolate pure populations of cardiomyocytes as identified by immunohistochemical staining of sarcomeric proteins (cardiac uoponin and myosin heavy chain). Isolated fetal pig cardiomyocytes show a limited potential for proliferation postisolation, consistent with what others have shown for fetal cardiomyocytes from other species. Culture conditions for growth, maintenance and contractility of cardiomyocytes were established. Cultured fetal pig cardiomyocytes masked with F(ab')2 fragments of anti pig class I antibody were injected directly into mouse myocardium. Implanted masked cardiomyecytes formed stable grafts over 30 day period, as evidenced by PRE (pig repeating element) in situ hybridization and PCR-PRE techniques.
HUMAN ENDOTHELIAL CELLS SHOW DIPI:P.:RENTIATED FUNCTIONS AFTER IN VITRO SEEDING ON eFIYE Jansson K, Bengtsson L, Hacgerstrand A; Stockholm SWEDEN
3.10
IMPROVED SURVIVAL IN ANHEPATIC RAT BY TRANSPLANTED HEPATOCYTES Lorenzo M, Angrisunl L, Di Prisco B, Cuomo 1l, Tesanro B; Naples Italy Aim of this study is to evaluate if hepatocytes U'ansplanted into the spleen may improve the survival in anhepatic rat, with any kind of concomitant pharmacologic support. A total hepntectomy was performed with an original method in which the liver was replaced by an heterologous
vasc~ar prosthe~ that joins the s~rahepaac cave. the subhepadc cuva and the portal vein. For all experiments male Sprague-Dawley rats (250-350 gr) were used. Hepatocytes (4x107) were transplanted by direct injection into the splenic parenchyma. Rats were allocated in the following groups: Group A (n=I0) that underwent a total hepatectomy only (control group); Group B (n=10), Group C (n=10) and Group D (n=10), that underwent hepatocyte transplantation respectively I, 2, and 9 months before of total hepatectomy. Statistical analysis was performed by means of Fisher's exact test and Student t-test. Survival of anlaepntic rats was significantly improved only in Group D rats, respect to control group(75+18 Vs 32+15; p<0.001). This fact indicate the need for hepatocytes to proliferate and colonize the engrafted organ. This result can be considered an useful tool to study the possibility to use hepatocyte trannsplantation as a bridge for orthotopic liver transplantation.
23