Effect of long-term culture on the expression of antigens and adhesion molecule in single porcine pancreatic endocrine cells.

BACKGROUND: Changes in the expression of galactose (Gal) alpha1-3Gal, swine lymphocyte antigen (SLA) class II and intracellular adhesion molecule (ICAM)-1 of single porcine pancreatic endocrine (PE) cells during the culture period were investigated. METHODS: Cultured porcine PE-cells were fixed in 10% buffered formalin for histological evaluation. At 1, 3, 6, 9 and 12 weeks of culture, mRNA was obtained from porcine PE-cells so that the expression of SLA class II and ICAM-1 genes could be examined by reverse transcriptase-polymerase chain reaction. RESULTS: The rates of Galalpha1-3Gal and SLA class II-positive cells did not decrease during the culture period, but the rates of Galalpha1-3Gal and SLA class II strongly positive cells significantly decreased. ICAM-1-positive cells were scarcely observed during the culture period. SLA class II and ICAM-1 mRNAs were detected at 1 and 3 weeks of culture, but were not detected after 6 weeks of culture. CONCLUSIONS: These results suggest that partial reduction in the expression of these antigens could be obtained by a long-term culture.

Prevalence of porcine endogenous retrovirus in domestic pigs in Japan and its potential infection in dogs xenotransplanted with porcine pancreatic islet cells.

The prevalence of porcine endogenous retrovirus (PERV) proviral DNA among various pig breeds raised in Japan was investigated by polymerase chain reaction (PCR). Moreover, potential infection of PERV was investigated by PCR and reverse transcriptase-polymerase chain reaction (RT-PCR) in experimentally induced diabetic dogs (n=5) implanted with the diffusion chamber type bio-artificial endocrine pancreas (Bio-AEP) containing porcine pancreatic endocrine (PE) cells. No immunosuppressant was used after the transplantation. PERV gag, pol, env-A and env-B genes were detected in any pigs examined. In two of three Landrace breeds, env-C gene was absent. PERV proviral DNAs and viral RNAs were also detected from the cultured porcine PE-cells. In the peripheral blood mononuclear cells and the spleen obtained at 6, 30, 32, 36, 79 weeks of xenotransplantation in dogs, however, no evidence of microchimerism, infection and viremia were confirmed. These results suggested that the risk of PERV infection through xenotransplantation of Bio-AEP containing porcine islet cells without immunosuppressants may be quite low.

Effect of adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness in the cultured adult porcine endocrine pancreas: a preliminary study.

The effect of either adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness was investigated in adult porcine pancreatic endocrine (PE) cells that were cultured for a longer term in vitro. Six different types of molecules--laminin, fibronectin, poly-L-lysine (PLL), type I collagen, gelatin, and Matrigel--were used. Approximately 2.0 x 10(5) cells per dish of each molecule type were cultured for 4 weeks. In the laminin group, the insulin accumulation was maintained at a significantly higher level than in the control group at 4 weeks of culture, and glucose-stimulated insulin secretion and the insulin-positive rate were also higher than in the control group. In the Matrigel group, islet-like cell clusters were formed, but insulin accumulation rapidly decreased at 3-4 weeks of culture. A large number of PE cells attached tightly and spread in the fibronectin group until the fourth week of culture, but their function was not better than those in the control group. In the PLL and gelatin groups, the PE cell function was not significantly different from that of the control group. In the type I collagen group, insulin secretion was inferior to that of the other groups. The results of this study suggest that laminin is the most suitable extracellular matrix for the long-term culture preservation of PE cells.

Xenotransplantation of porcine pancreatic endocrine cells to total pancreatectomized dogs.

Xenotransplantation of porcine pancreatic endocrine (PE) cells in a diffusion chamber, a bioartificial endocrine pancreas (Bio-AEP), was conducted to total pancreatectomized dogs. Six pancreatectomized dogs were divided into two groups of 3 dogs each. In three dogs of the control group, exogenous insulin was administered twice a day for 30 weeks to maintain fasting blood glucose (FBG) levels within the normal range. The remaining three dogs were implanted with Bio-AEPs (implantation group), in addition to daily insulin administration. In the implantation group, Bio-AEPs containing 1.3 to 1.8 x 10(7) cells per kg of body weight of the recipient were implanted without fixation into the abdominal cavity. In the control group, exogenous insulin requirements did not decrease during the experimental period, whereas it significantly decreased for a certain period (3, 11, 17 weeks) after implantation in all implanted dogs. In the implantation group, laparotomy was performed after FBG and the exogenous insulin requirement increased again and Bio-AEPs were removed. Two Bio-AEPs were completely destroyed, and the remaining one was encapsulated by thin fibrous tissue. In this dog, effusion was present within the capsule, but the Bio-AEP was not destroyed. Histopathologically, the necrosis, presumably caused by hypoxia, of the PE-cells was observed on transmission electron microscopy. In conclusion, Bio-AEP could function for a certain period after implantation in this study. However, more preclinical researches should be needed to apply this technique for the treatment of diabetic dogs.

Comparative study of adult porcine pancreatic endocrine cell preparation using a technique of multiple injections and pancreatic duct cannulation without a proteolytic enzyme.

INTRODUCTION: A method for isolation and primary monolayer culture of pancreatic endocrine (PE) cells from the porcine pancreas has already been established. It is very important for the PE cell preparation to expand the pancreas to separate PE cells from acinar cells. For this purpose, we developed a pancreatic injection system. AIM: To compare two pancreatic injection methods: perfusion from an accessory pancreatic duct (cannulation method) and the traditional pancreatic tissue injection method (multiple injection method). RESULTS: A comparison of the results of the two methods revealed that the PE cell yield was significantly higher with the cannulation method (2.97 +/- 0.59 x 10(7) cells per pancreas) than with the multiple injection method (0.89 +/- 0.15 x 10(7) cells per pancreas) (p < 0.0001). The number of dithizone-positive cells was significantly higher with the cannulation method (1.64 +/- 0.36 x 10(7) cells per pancreas) than with the multiple injection method (0.36 +/- 0.09 x 10(7) cells per pancreas) (p < 0.0001). The number of adhesion cells after 7 days of culture following isolation was higher with the cannulation method (1.07 +/- 0.26 x 10(7) cells per pancreas) than with the multiple injection method (0.36 +/- 0.03 x 10(7) cells per pancreas) (p < 0.0001). The glucose stimulation index of insulin secretion was higher with the cannulation method than with the multiple injection method (p < 0.01). CONCLUSION: These results indicate that pancreatic duct perfusion is useful for obtaining a high yield of PE cells from porcine pancreases.