8 research outputs found
Stimulation of vascularization of a subcutaneous scaffold applicable for pancreatic islet-transplantation enhances immediate post-transplant islet graft function but not long-term normoglycemia
Nephrolog
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Improved in vitro function of islets using small intestinal submucosa
Transplantation of human pancreatic islets has been demonstrated to be a viable alternative to exogenous insulin therapy for diabetes mellitus. However, optimum results require transplantation of islets from two to three pancreas donors after a minimum number of days in culture. This implies that a substantial part of the transplanted islet mass may be nonfunctional. This study investigates the ability of an optimized technique to retain islet function using porcine-derived small intestinal submucosa (SIS) during in vitro culture.
Groups of purified human islets were cultured for 3 weeks in modified standard islet culture conditions of CMRL = 1066 tissue culture medium supplemented with 25 mmol/L HEPES, penicillin/streptomycin, and a commercial insulin-transferin-selenium (ITS) supplement. Islets (50 to 200 IE/condition; n = 5 preparations) were cultured in plates containing noncoated Biopore membrane inserts alone, or on inserts that had been covered with SIS. Function was assessed by static incubation with low (4 mmol/L), or high (20 mmol/L) glucose at the end of each week. Glucose-stimulated release of human insulin was measured by radioimmunoassay (Linco, St. Charles, Missouri). Remaining islets were stained and evaluated visually. Neither culture condition resulted in significantly different basal secretion until week 3 (
P = .05). However, by the end of week 2 and for the duration of the experiment thereafter, SIS-treated islets exhibited a higher SI (
P < .05). At the end of the experiment, islets cultured on the SIS exhibited excellent morphology, with greater than 90% staining positive with Dithizone. Islets cultured on the inserts alone lost their initial morphology, becoming “loose” in appearance. The results of this study indicate that SIS enables enhanced function of islets in vitro as compared to non-SIS supported culture conditions
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Enhanced recovery of cryopreserved islets using SIS
Although cryopreservation of pancreatic islets would add flexibility to transplantation, the recoveries are only 60% to 90% and function is decreased. Islets are multicellular structures ∼50 to 250 μm in diameter organized into a network of cells and vascular channels. Due to this complexity, islets are more susceptible to damage during cryopreservation than an individual cell. This study investigated porcine small intestinal submucosa (SIS) as a matrix to support islets recovery and function post-thaw. Groups of frozen/thawed human islets (150 IE/condition;
n = 4 preparations) were cultured for 5 weeks in plates containing noncoated Biopore membrane inserts alone or inserts covered with SIS. Islets were placed directly on the insert post-thaw (SIS
1), or cultured overnight in standard plates, washed, and then transferred to the SIS (SIS
2). Function was assessed by determining glucose-stimulated release of insulin, which was measured by radioimmunoassay. Analysis of basal insulin secretion showed time and treatment to be significantly different (
P = .0043 and
P = .0123, respectively) but without an interaction (
P > .05). The two SIS treatments were not significantly different (
P > .05); however, both SIS
1 and SIS
2 were significantly different from controls (
P = .0108 and
P = .0420, respectively). Similar results were obtained for stimulation indices; time and treatment were significantly different (
P = .0161 and
P = .0264, respectively) but not an interaction (
P > .05). The two SIS treatments were not significantly different (
P = .05); however, both SIS
1 and SIS
2 differed from controls (
P = .0248 and
P = .0407, respectively). The results indicate that SIS enables frozen-thawed islets to exhibit superior post-thaw function compared with a non-SIS–supported condition
Peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells
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