Small intestinal submucosa improves islet survival and function in vitro culture.
BACKGROUND
Most centers maintain isolated human islet preparations in tissue culture to improve the safety as well as the practicality of islet transplantation. However, maintaining viability and recovery of islets remains a challenge. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell-matrix relationship seems to be necessary to sustain the structure and function of differentiated islets. Small intestinal submucosa (SIS), a natural ECM, is well known to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate recovery and function of isolated rat pancreatic islets during in vitro culture with SIS.


METHODS
Pancreatic islets isolated from Wistar rats following intraductal collagenase distension, mechanical dissociation, and EuroFicoll purification were cultured in plates coated with multilayer SIS (SIS-treated group) or without (standard cultured group) for 7 or 14 days in an islet culture media of RPMI 1640 (Gibco). The islets from both experimental groups were stained and counted with dithizone. Islet recovery following culture was determined by the ratio of counts after culture to the yield of islets immediately following islet isolation. The viability of the islets was assessed by a glucose challenge test with low glucose (2.7 mmol/L), high glucose (16.7 mmol/L), and high glucose solution supplemented with 50 micromol/L 3-isobutyl-1-methylxanthine solution. The apoptosis of islet cells was measured by relative quantification of histone-complexed DNA fragments by using enzyme-linked immunosorbent assay.


RESULTS
After 7 or 14 days of in vitro tissue culture, the recovery in SIS-treated islets group was about double of that cultured in the plates without SIS coating. In the SIS-treated group, there was no significant difference between the short- and the long-term periods of culture (95.8%+/-1.0% vs 90.8%+/-1.5%, P>.05). Following incubation with high glucose (16.7 mmol/L) solution, the insulin secretion in the SIS-treated group showed a greater increase than the control group after 14 days of culture (20.7+/-1.1 mU/L vs 11.8+/-1.1 mU/L, P<.05). When islets were placed in the high glucose solution containing IBMX, the stimulated insulin secretion was more increased in the SIS-treated than in the control group despite the duration of the culture. The calculated stimulation index of SIS-treated group was about two to three times greater than the control group. In addition, the stimulation index of the SIS-treated group remained constant regardless of short-term versus long-term culture (9.5+/-0.2 vs 10.2+/-1.2, P>.05). Much less apoptosis of islet cells occurred in the SIS-treated than in the control group.


CONCLUSIONS
Coculture of isolated rat islets with native sheetlike small intestinal submucosa seemed to build an ECM for islets providing possible biotrophic and growth factors that promote the recovery and subsequent function of islets.
Xiaohui
T
T
Department of Renal Transplantation, First Hospital of the Xi'an Jiao Tong University, Xi'an, ShannXi, China, People's Republic of China.
Wujun
X
X
Xiaoming
D
D
Xinlu
P
P
Yan
T
T
Puxun
T
T
Xinshun
F
F
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation; D002470:Cell Survival; D017364:Collagenases; D005109:Extracellular Matrix / Q000502:physiology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007413:Intestinal Mucosa / Q000502:physiology*; D007421:Intestine, Small; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D023421:Models, Animal; D051381:Rats; D017208:Rats, Wistar
D007328:Insulin; D017364:Collagenases; D005947:Glucose