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Cryopreservation of fish islets: The effect on function and islet xenograft survival
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2.03
Cell Transplantation • Volume 5, Number 5S-2, 1996 CRYOPRESERVATION OF FISH ISLETS: THE EFFECT ON FUNCTION AND ISLET XENOGRAFT SURVIVAL O'Hali W, Pohajdak B, Yang H, Gross M, Wright JR Jr; Halifax CANADA
2.04
CRYOPRESERVED HUMAN ISLETS IN VITRO ALTERATION OF INSULIN AND PRO-INSULIN SECRETION. Socci C, Piemonti L, Bertuzzi F, De Nittis P, Monti L, Taglietti MV, Magistretti P, Nano R, Di Carlo V, Pozza G. Milano, ITALY. The metabolic effects of cryopreservation on isolated pancreatic islet function was investigated using six consecutive preparations not suitable for transplantation in human. After purification on discontinuous EuroFicoll gradients, islets were cryopreserved according to Rajotte method until nucleation; then the tubes were put into a computerized device to decrease the temperature from -9 to -50°C at a rate of 0.3 °C/rain. After a preliminary thawing in a warm bath, the material was immersed in 1 liter of M199 plus sucrose at 4°C. In vitro function was evaluated two days after isolation or thawing by perifnsion of fresh and cryopresexved islets. Insulin and pro-insulin secretion was investigated after stimulation with four different secretagogues: Glucose 16.7 raM; Glucose 16.7 mM plus IBMX 0.1 raM; Arginine 10raM end Tolbutamide 100 laM. Insulin and insulin like molecules were detected by RIA method. True insulin was assessed with a Microparticle Enzyme Immuno Assay method (MEIA). Proinsulin amount was obtained subtracting the levels of insulin indicated by MEIA from those of RIA. Basal insulin secretion was found increased in cryopreserved islet~ both for RIA and MEIA assessment (fresh vs cryo: RIA 4 vs 6.,~ pg/islet/min, ns; MEIA 2.43 vs 4.15 pg/islet/min, 13<0.05). Basa~ proinsulin secretion was also found increased (fresh vs cryo 1.58 vs 2.26 ng/islet/min). After stimulation, cryopreserved islets produced mor~ proinsulin than fresh islets for the four secretagogues studied~ with a significant statistical difference for arginine (AUC fresh v~ cryo: 21 vs 59 pg/islet/20min, p
2.06
THE INTRACELLULAR ATP CONTENT OF FRESH AND CULTURED HUMAN ISLETS ISOLATED FROM DIFFERENT DONORS. Brandhorst D, Brandhorst H, Hering BJ, Federlin K, Bretzel RG, Giessen, GERMANY Observations in experimental heart, liver, kidney and pancreas transplantation indicated that graft function and survival correlates significantly with ATP content of transplanted tissue. This study investigates for the first time the intracellular ATP content of isolated human islets. Quantified samples of freshly isolated (digestion-filtration, continuous gradient purification) and cultured (22°C, CMRL+10% FCS) islets of consecutively processed human pancreata were shock frozen and stored at -196°C until subsequent determination of ATP (luminometric Luciferin-Luciferase-reaction) and islet protein (IP). Each experiment was performed in quadruplicate and considered as one single observation ~). The ATP content (pg/lag IP) was evaluated for freshly isolated and subsequently 5±1d cultured islets (n=10) or after incubation in different glucose concentrations of (a) 500, (b) 750, (c) 1000 and (d) 2000 mg/l for 9±ld (n=6). The ATP content of freshly isolated human islets was 130.4±53.4 pg/lag IP (~±SEM). After culture ATP content was increased to 265.5:t:113.3 pg/lag IP corresponding to 204.2:~41.5% (p<0.05, Wilcoxon-test). The coefficient of variation for fresh and cultured islets was 129.5% and 135.0%, respectively. The relative ATP content (cultured/fresh) per lag IP after culture in different glucose concentrations was (a) 77.9±18.1% (p<0.05 vs b, c); (b) 116.4±20.1%; (c) 145.0±33.4% and (d) 94.7±22.4% (p<0.05 vs b, c). The present dam indicate that: (1) the ATP content of freshly isolated human islets varies enormously; (2) intraislet ATP levels increase significantly during 22°C culture suggesting that the capacity to produce ATP is maintained (3) ATP synthesis is most effective at a glucose concentration of 750 and 1000 mg/l.
This study examines whether tilapia Brockmann bodies (BBs), large anatomically discrete pancreatic islets in certain teleost fish, can be cryopreserved and thawed without loss of function and whether cryopreservation prolongs BB xenograft survival. BBs were harvested, cultured overnight, and then cryopreserved using Rajotte's slow cooling rat islet protocol. After 3-14 days of storage, the BBs were rapidly thawed and cultured overnight. Fluoroscein diacetate/ethidium bromide staining demonstrated a mixture of viable and nonviable cells post-thaw. BBs were transplanted under the kidney capsule of STZ-diabetic (blood glucose levels > 400 mg/dl) athymic nude mice. Normalizing non-fasting blood glucose in 6 of 8; these mice maintained blood glucose levels in the range of 100-150 mg/dl for > 30 days. Graft nephrectomy, performed on day 31, confirmed graft function. Sections of grafts and native pancreata, stained with H&E and for insulin, were examined histologically. All mice showed abundant well-granulated islets under the renal capsules. Cryopreserved BBs were also transplanted to STZ-diabetic euthymic balb/c mice. Rejection was defined as two successive non-fasting blood glucose levels > 200 mg/dl. Gratt survival times (GST) for control and cryopreserved BBs were 6,7,7,8,8, and 8 days vs 6,7,8,10,11, and 12 days, respectively (t=1.63, df=10, p=0.133). We conclude that tilapia BBs can be cryopreserved using a standard mammalian islet protocol and that cryopreserved BBs will maintain long-term function when transplanted to STZdiabetic nude mice. Cryopreservation does not prolong BBs xenograft survival.
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CELL CULTURE PREPARATION OF HUMAN PARATHYROID CELLS FOR ALLOTRANSPLANTATION WITHOUT IMMUNOSUPPRESSION Wo~iewicz B, Migaj M. Giera B, Prokurat A, Tolloczko T, Sawieki A, Nawrot I, G6rski A, Zabitkowska T,Warsaw,PL Kossakowska AE; Calgary, Alberta. In 1989 we have observed that donors hyperplnstic parathyroid grain had shown increased number of CD3, CD4, CI~, CD68, CD31 positive ceils and high expression of HLA class I and class II antigens when compared with normal control.We have decided to isolate a pure population of end~rine parathyroid cells by the way of tissue calULre.Donors parathyroid received during surgery due to secondary hypetparathyroidism were carefuly assessed for structural and fun~ional histoiogic abnormalities using a panel of the following anUlx~dies anti- see above- and CD22, Fibroblast,PTI~ HLA class I ,class H anlbodies and APAAP methods. Similar studies were perform on ceils growing in vitrO. Sterile fresh or ctyopreserved about 15 fragments less than Imm together with cell suspension obtained during mincing of the parathyroid were cultured in standard 25mi Falcon flask and standard Eagle medium with I0% fetal calf serum and Gentamycin 5mg/100mi with adding of 0,02M L-glutamine. The culture medium were removed every 3-5 days and PTH -C terminal reached in superoatant from 10-38 ng per mi. During about 6 weeks of primary culture a monolayer growth of cultured ceils covered total bottom of area of flask (--about 2-3 millions cells ).Before storing of cells vital staining and stmctmal /electron microscopic/ and functional h~aenotyping of cell population/ were performed. In 35 patients with surgical hypopamthyroidism ~tation was succesfid Survival time of cells transplanted in forearm was in the range 3-18 months.
2.03
Cell Transplantation • Volume 5, Number 5S-2, 1996 CRYOPRESERVATION OF FISH ISLETS: THE EFFECT ON FUNCTION AND ISLET XENOGRAFT SURVIVAL O'Hali W, Pohajdak B, Yang H, Gross M, Wright JR Jr; Halifax CANADA
2.04
CRYOPRESERVED HUMAN ISLETS IN VITRO ALTERATION OF INSULIN AND PRO-INSULIN SECRETION. Socci C, Piemonti L, Bertuzzi F, De Nittis P, Monti L, Taglietti MV, Magistretti P, Nano R, Di Carlo V, Pozza G. Milano, ITALY. The metabolic effects of cryopreservation on isolated pancreatic islet function was investigated using six consecutive preparations not suitable for transplantation in human. After purification on discontinuous EuroFicoll gradients, islets were cryopreserved according to Rajotte method until nucleation; then the tubes were put into a computerized device to decrease the temperature from -9 to -50°C at a rate of 0.3 °C/rain. After a preliminary thawing in a warm bath, the material was immersed in 1 liter of M199 plus sucrose at 4°C. In vitro function was evaluated two days after isolation or thawing by perifnsion of fresh and cryopresexved islets. Insulin and pro-insulin secretion was investigated after stimulation with four different secretagogues: Glucose 16.7 raM; Glucose 16.7 mM plus IBMX 0.1 raM; Arginine 10raM end Tolbutamide 100 laM. Insulin and insulin like molecules were detected by RIA method. True insulin was assessed with a Microparticle Enzyme Immuno Assay method (MEIA). Proinsulin amount was obtained subtracting the levels of insulin indicated by MEIA from those of RIA. Basal insulin secretion was found increased in cryopreserved islet~ both for RIA and MEIA assessment (fresh vs cryo: RIA 4 vs 6.,~ pg/islet/min, ns; MEIA 2.43 vs 4.15 pg/islet/min, 13<0.05). Basa~ proinsulin secretion was also found increased (fresh vs cryo 1.58 vs 2.26 ng/islet/min). After stimulation, cryopreserved islets produced mor~ proinsulin than fresh islets for the four secretagogues studied~ with a significant statistical difference for arginine (AUC fresh v~ cryo: 21 vs 59 pg/islet/20min, p
2.06
THE INTRACELLULAR ATP CONTENT OF FRESH AND CULTURED HUMAN ISLETS ISOLATED FROM DIFFERENT DONORS. Brandhorst D, Brandhorst H, Hering BJ, Federlin K, Bretzel RG, Giessen, GERMANY Observations in experimental heart, liver, kidney and pancreas transplantation indicated that graft function and survival correlates significantly with ATP content of transplanted tissue. This study investigates for the first time the intracellular ATP content of isolated human islets. Quantified samples of freshly isolated (digestion-filtration, continuous gradient purification) and cultured (22°C, CMRL+10% FCS) islets of consecutively processed human pancreata were shock frozen and stored at -196°C until subsequent determination of ATP (luminometric Luciferin-Luciferase-reaction) and islet protein (IP). Each experiment was performed in quadruplicate and considered as one single observation ~). The ATP content (pg/lag IP) was evaluated for freshly isolated and subsequently 5±1d cultured islets (n=10) or after incubation in different glucose concentrations of (a) 500, (b) 750, (c) 1000 and (d) 2000 mg/l for 9±ld (n=6). The ATP content of freshly isolated human islets was 130.4±53.4 pg/lag IP (~±SEM). After culture ATP content was increased to 265.5:t:113.3 pg/lag IP corresponding to 204.2:~41.5% (p<0.05, Wilcoxon-test). The coefficient of variation for fresh and cultured islets was 129.5% and 135.0%, respectively. The relative ATP content (cultured/fresh) per lag IP after culture in different glucose concentrations was (a) 77.9±18.1% (p<0.05 vs b, c); (b) 116.4±20.1%; (c) 145.0±33.4% and (d) 94.7±22.4% (p<0.05 vs b, c). The present dam indicate that: (1) the ATP content of freshly isolated human islets varies enormously; (2) intraislet ATP levels increase significantly during 22°C culture suggesting that the capacity to produce ATP is maintained (3) ATP synthesis is most effective at a glucose concentration of 750 and 1000 mg/l.
This study examines whether tilapia Brockmann bodies (BBs), large anatomically discrete pancreatic islets in certain teleost fish, can be cryopreserved and thawed without loss of function and whether cryopreservation prolongs BB xenograft survival. BBs were harvested, cultured overnight, and then cryopreserved using Rajotte's slow cooling rat islet protocol. After 3-14 days of storage, the BBs were rapidly thawed and cultured overnight. Fluoroscein diacetate/ethidium bromide staining demonstrated a mixture of viable and nonviable cells post-thaw. BBs were transplanted under the kidney capsule of STZ-diabetic (blood glucose levels > 400 mg/dl) athymic nude mice. Normalizing non-fasting blood glucose in 6 of 8; these mice maintained blood glucose levels in the range of 100-150 mg/dl for > 30 days. Graft nephrectomy, performed on day 31, confirmed graft function. Sections of grafts and native pancreata, stained with H&E and for insulin, were examined histologically. All mice showed abundant well-granulated islets under the renal capsules. Cryopreserved BBs were also transplanted to STZ-diabetic euthymic balb/c mice. Rejection was defined as two successive non-fasting blood glucose levels > 200 mg/dl. Gratt survival times (GST) for control and cryopreserved BBs were 6,7,7,8,8, and 8 days vs 6,7,8,10,11, and 12 days, respectively (t=1.63, df=10, p=0.133). We conclude that tilapia BBs can be cryopreserved using a standard mammalian islet protocol and that cryopreserved BBs will maintain long-term function when transplanted to STZdiabetic nude mice. Cryopreservation does not prolong BBs xenograft survival.
2.05
CELL CULTURE PREPARATION OF HUMAN PARATHYROID CELLS FOR ALLOTRANSPLANTATION WITHOUT IMMUNOSUPPRESSION Wo~iewicz B, Migaj M. Giera B, Prokurat A, Tolloczko T, Sawieki A, Nawrot I, G6rski A, Zabitkowska T,Warsaw,PL Kossakowska AE; Calgary, Alberta. In 1989 we have observed that donors hyperplnstic parathyroid grain had shown increased number of CD3, CD4, CI~, CD68, CD31 positive ceils and high expression of HLA class I and class II antigens when compared with normal control.We have decided to isolate a pure population of end~rine parathyroid cells by the way of tissue calULre.Donors parathyroid received during surgery due to secondary hypetparathyroidism were carefuly assessed for structural and fun~ional histoiogic abnormalities using a panel of the following anUlx~dies anti- see above- and CD22, Fibroblast,PTI~ HLA class I ,class H anlbodies and APAAP methods. Similar studies were perform on ceils growing in vitrO. Sterile fresh or ctyopreserved about 15 fragments less than Imm together with cell suspension obtained during mincing of the parathyroid were cultured in standard 25mi Falcon flask and standard Eagle medium with I0% fetal calf serum and Gentamycin 5mg/100mi with adding of 0,02M L-glutamine. The culture medium were removed every 3-5 days and PTH -C terminal reached in superoatant from 10-38 ng per mi. During about 6 weeks of primary culture a monolayer growth of cultured ceils covered total bottom of area of flask (--about 2-3 millions cells ).Before storing of cells vital staining and stmctmal /electron microscopic/ and functional h~aenotyping of cell population/ were performed. In 35 patients with surgical hypopamthyroidism ~tation was succesfid Survival time of cells transplanted in forearm was in the range 3-18 months.