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Experimental xenotransplantation of fresh isolated and cryopreserved pig hepatocytes-biochemical and morphological study
Cell Transplantation, Vol. 5, No. 5S-2, pp. 37~-3, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0963-6897/96 $15.00 + .00 ELSEVIER
PII S0963-6897(96)00164-9
Transplant Sites and Techniques Category 5
5.01
EXPERIMENTAL XENOTRANSPLANTATIONOF FRESH ISOLATEDAND CRYOPRESERVEDPIGHEPATOCYTES-BIOCHEMICALAND MORPHOLOGICALSTUDY ]Pspldw A, Arkadopoalos N, Kusmpanngiotou G, Tbeodorekis K, Peveretos P, Golematis & Papadimitriou .I,Allures, OILImCE Orthotopic liver transplantation (OLTx) is an established therapeutic approach to fulminant liver failure (LF).Various methods have been developed for the temporary memhefic support of acute IF. Hepatocyto (Hc) ~x has many experimental applications The aim of this protuco[ was to assess the effects of our cryopresorvation storage method on the viability and funtion of Hc and compare the ability of fresh and cryopreservad stored Hc to support animals in severe toxic liver failure after being transplanted in the splenic pumuchyma and beneath the kidney capsule. Two female swine 18-20kg were used as l-k:donors Thirty Lewis rats weighing200-300 gr served as recipients Recipients were divided in 3 group~ Toxic acute liver fai!ure CTALF)was induced in all groups by a single dose of N-Dimethylonitmsomine (N-DM]qA, 20 mg/Kg IV). Group A (n--6) underwent induction of TALF.witbout further treatment Group B (n=12) underwent induction of TALF followed m 24h by ~ of I0° fresh isolated xeno*HcGroup C (n=]2) underwent induction of TALF followed in 24h by Tx of 10e cryopreserved xeno-H," Hc were prepared according to our modification of the Snglen tuchniq~ Viability was 9 2 ~ by the trypan blue exclusion test Following isolation were either transplanted or cxTopreserved.Hc were cryopreserved at -20°C for one month. All groups were treated with cyclusporin A (CsA) 20 mg/kg/day IV (days 0-15)and I0 mg/kg/day (days 15-30).All surviving mrs were euthanised at day 30. Survival rates at 30 days for groups A,B and C were 0~ 50~ and 4L6%respectively. Mean post transplant values do not differ significantly between groups B and C The AST and ALT values reached their maximum on days 2-3 post transplant and return to normal after day 10-12for group B and 12-16for group C The bili[ubin values reached their maximum on day 2 post Tx and returned to normal tftor day 20-28 for both groupx Mortality after N-DMNA administration correlated well with the extent of liver damage (hemorrhagic nen~lobular necrosis). The Liven of rats surviving more than 14 days in both groups B and C showed signs of regeucmtion of the normal architectur~ Aggregates of viable Hc were found in the spleen occupying 20-30~ of the splenic red pulp in groups B-C. Cords of He were observed under the kidney capsule in groups B and C Hc contained glucogen in group B (PAS stain) but not in group C Our cryopreservation and storage method reduced cell viability by 3Y& however our isolation method results in big humbert of cells and we can afford to lose some in the storing process. In terms of fuuctinn both fresh and cryopresorved Hc proved to be equally effective (no statistically significant differeDcoobserved).
5.02
D E V E L O P M E N T O F A L A P A R O S C O P I C A P P R O A C H FOR ISLET T R A N S P L A N T A T I O N P e p a l o l a Ap Bonatsos G, Birbas C, Toutouzas If., Goleraatis B, A t h e n s , ORBBCR Islet transplantation (Tx) prolong insulin independence a f t e r allo-Tx in humans. In o r d e r to p e r f o r m islet Tx, an open surgical procedure (laparotomy) is the necessary approach. T h e a i m of this e x p e r i m e n t a l study was to develop a technique of islet cell Tx using a laparo-endoscopic procedure. The laparoseepic surgery has shown advantages in comparison to open surgery as less postoperative complications and less hospitalization. In this e x p e r i m e n t a l protocol 12 f e m a l e swines (18-21 kg) were used (6 as donors of islets and 6 as recipients). In the group of donors, total p a n c r e a t e c t o m y was p e r f o r m e d and the islets w e r e isolated using the collagenase digestion technique (Sigma, Type XI, 1 mg/ml). Cell were purified on EureeollinsFiroll density gradients. The n u m b e r of isolated cells ranged f r o m >70,000 to ,80,000 a n d purity was g r e a t e r than 85%. In the group of recipients (normal animals), a f t e r establishing a pneumoperitoneum, three laparoscopic trocars w e r e placed. A mesenteric vein was catheterized and islets infused slowly. All animals were euthanised at d a y 5. Morphological examination c o n f i r m e d the implantation of islets in the portal system. In conclusion this preliminary experimental protocol describes a safe laparoscopic procedure for islet Tx and this is applicable in a variety of experimental protocols as well as in clinical practice.
PII S0963-6897(96)00164-9
Transplant Sites and Techniques Category 5
5.01
EXPERIMENTAL XENOTRANSPLANTATIONOF FRESH ISOLATEDAND CRYOPRESERVEDPIGHEPATOCYTES-BIOCHEMICALAND MORPHOLOGICALSTUDY ]Pspldw A, Arkadopoalos N, Kusmpanngiotou G, Tbeodorekis K, Peveretos P, Golematis & Papadimitriou .I,Allures, OILImCE Orthotopic liver transplantation (OLTx) is an established therapeutic approach to fulminant liver failure (LF).Various methods have been developed for the temporary memhefic support of acute IF. Hepatocyto (Hc) ~x has many experimental applications The aim of this protuco[ was to assess the effects of our cryopresorvation storage method on the viability and funtion of Hc and compare the ability of fresh and cryopreservad stored Hc to support animals in severe toxic liver failure after being transplanted in the splenic pumuchyma and beneath the kidney capsule. Two female swine 18-20kg were used as l-k:donors Thirty Lewis rats weighing200-300 gr served as recipients Recipients were divided in 3 group~ Toxic acute liver fai!ure CTALF)was induced in all groups by a single dose of N-Dimethylonitmsomine (N-DM]qA, 20 mg/Kg IV). Group A (n--6) underwent induction of TALF.witbout further treatment Group B (n=12) underwent induction of TALF followed m 24h by ~ of I0° fresh isolated xeno*HcGroup C (n=]2) underwent induction of TALF followed in 24h by Tx of 10e cryopreserved xeno-H," Hc were prepared according to our modification of the Snglen tuchniq~ Viability was 9 2 ~ by the trypan blue exclusion test Following isolation were either transplanted or cxTopreserved.Hc were cryopreserved at -20°C for one month. All groups were treated with cyclusporin A (CsA) 20 mg/kg/day IV (days 0-15)and I0 mg/kg/day (days 15-30).All surviving mrs were euthanised at day 30. Survival rates at 30 days for groups A,B and C were 0~ 50~ and 4L6%respectively. Mean post transplant values do not differ significantly between groups B and C The AST and ALT values reached their maximum on days 2-3 post transplant and return to normal after day 10-12for group B and 12-16for group C The bili[ubin values reached their maximum on day 2 post Tx and returned to normal tftor day 20-28 for both groupx Mortality after N-DMNA administration correlated well with the extent of liver damage (hemorrhagic nen~lobular necrosis). The Liven of rats surviving more than 14 days in both groups B and C showed signs of regeucmtion of the normal architectur~ Aggregates of viable Hc were found in the spleen occupying 20-30~ of the splenic red pulp in groups B-C. Cords of He were observed under the kidney capsule in groups B and C Hc contained glucogen in group B (PAS stain) but not in group C Our cryopreservation and storage method reduced cell viability by 3Y& however our isolation method results in big humbert of cells and we can afford to lose some in the storing process. In terms of fuuctinn both fresh and cryopresorved Hc proved to be equally effective (no statistically significant differeDcoobserved).
5.02
D E V E L O P M E N T O F A L A P A R O S C O P I C A P P R O A C H FOR ISLET T R A N S P L A N T A T I O N P e p a l o l a Ap Bonatsos G, Birbas C, Toutouzas If., Goleraatis B, A t h e n s , ORBBCR Islet transplantation (Tx) prolong insulin independence a f t e r allo-Tx in humans. In o r d e r to p e r f o r m islet Tx, an open surgical procedure (laparotomy) is the necessary approach. T h e a i m of this e x p e r i m e n t a l study was to develop a technique of islet cell Tx using a laparo-endoscopic procedure. The laparoseepic surgery has shown advantages in comparison to open surgery as less postoperative complications and less hospitalization. In this e x p e r i m e n t a l protocol 12 f e m a l e swines (18-21 kg) were used (6 as donors of islets and 6 as recipients). In the group of donors, total p a n c r e a t e c t o m y was p e r f o r m e d and the islets w e r e isolated using the collagenase digestion technique (Sigma, Type XI, 1 mg/ml). Cell were purified on EureeollinsFiroll density gradients. The n u m b e r of isolated cells ranged f r o m >70,000 to ,80,000 a n d purity was g r e a t e r than 85%. In the group of recipients (normal animals), a f t e r establishing a pneumoperitoneum, three laparoscopic trocars w e r e placed. A mesenteric vein was catheterized and islets infused slowly. All animals were euthanised at d a y 5. Morphological examination c o n f i r m e d the implantation of islets in the portal system. In conclusion this preliminary experimental protocol describes a safe laparoscopic procedure for islet Tx and this is applicable in a variety of experimental protocols as well as in clinical practice.