Small intestinal submucosa improves islet survival and function during in vitro culture.
OBJECTIVE
To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa (SIS).


METHODS
Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS (SIS-treated group) or without multilayer SIS (standard cultured group) for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge test with low (2.7 mmol/L) and high glucose (16.7 mmol/L) solution supplemented with 50 mmol/L 3-isobutyl-1-methylxanthine (IBMX) solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA.


RESULTS
After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS-treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture (95.8+/-1.0% vs 90.8+/-1.5%, P>0.05). When incubated with high glucose (16.7 mmol/L) solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture (20.7+/-1.1 mU/L vs 11.8+/-1.1 mU/L, P<0.05). When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SIS-treated group than in control group. Calculated stimulation index of SIS-treated group was about 23 times of control group. In addition, the stimulation index of SIS-treated group remained constant regardless of short- and long-term periods of culture (9.5+/-0.2 vs 10.2+/-1.2, P>0.05). Much less apoptosis of islet cells occurred in SIS-treated group than in control group after the culture.


CONCLUSIONS
Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and provide possible biotrophic and growth factors that promote the recovery and subsequent function of islets.
Tian
Xiao-Hui
XH
Center of Renal Transplantation, The First Hospital of Xioan Jiaotong University, Xioan 710061, Shaanxi Province, China.
Xue
Wu-Jun
WJ
Ding
Xiao-Ming
XM
Pang
Xin-Lu
XL
Teng
Yan
Y
Tian
Pu-Xun
PX
Feng
Xin-Shun
XS
D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D018920:Coculture Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007413:Intestinal Mucosa / Q000502:physiology*; D007421:Intestine, Small / Q000502:physiology; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D046509:Tissue Culture Techniques
D007328:Insulin