Improved Islet Survival and In Vitro Function Using Small Intestinal Submucosa J.R.T. Lakey, J. Troendle, M.A.J. Zieger, W.A. Geary, S. Voytek, and J.K. Critser I N VIVO proliferation of isolated pancreatic islets has become an area of great interest given the scarcity of clinical islet donors and the islet mass requirements for clinical islet transplantation. Small intestinal submucosa (SIS) is a naturally occurring extracellular matrix derived from the intestine which has been investigated as a source of biotrophic factors that promote wound healing, tissue remodeling, and cell growth. 1 It was the aim of this study to evaluate the recovery and in vitro function of isolated canine pancreatic islets cocultured with a solubilized SIS. MATERIALS AND METHODS Pancreatic islets were isolated from mongrel dogs using a standard surgical pancreas harvest followed by intraductal collagenase di- gestion, mechanical dissociation, and EuroFicoll purification. 2,3 Groups of purified islets were cultured in a humidified atmosphere of 5% air and 5% CO 2 for a period of 48 hours in standard islet culture conditions of CMRL 1066 tissue culture media (Gibco) which had been supplemented with 25 mmol/L HEPES, penicillin/ streptomycin, and either 10% heat-inactivated fetal calf serum (FCS, Gibco) or 10% solubilized SIS material. The mean recovery of islets following 48-hour culture was determined by staining with dithizone and sizing duplicate aliquots using established protocols. 4 Viability was assessed using the static incubation with low glucose (2.8 mmol/L), high glucose (20 mmol/L) and high glucose solution supplemented with 50 m IBMX for a period of 2 hours at 37°C. RESULTS AND DISCUSSION From a consecutive series of six culture experiments, we observed a significantly higher recovery of canine islets following 48-hour in vitro tissue culture with SIS-supple- mented media compared with the control group of islets cultured in standard conditions. A total of 84.5  2.9% (mean  SEM) of the islet mass was recovered in the SIS-cultured group compared with 64.7  4.5% from the control group cultured in FCS (P  .05, paired t test, n = 6). The proportion of islets with a mean diameter 150 m in diameter increased from 24% to 31% in the SIS-treated group, whereas the same proportion decreased to 18% from 22% in the control FCS-treated group. Islets from the SIS-treated group exhibited a significantly higher insulin response to the high glucose stimulus than islets cultured in the standard FCS cultured solution. The calculated stimu- lation index (SI, insulin secretion during high glucose + IBMX divided by basal insulin secretion during low glucose incubation) was 12.3  3.4 for the SIS-treated group compared with 5.6  1.8 for the standard cultured group (P  .05). Immunocytochemical staining of histologic sam- ples collected following the culture identified insulin and glucagon-secreting cells in both the SIS and FCS-treated groups. An immunocytochemical marker for cell prolifera- tion, proliferating cell nuclear antigen (PCNA), identified positive cells within the SIS cocultured group at a higher frequency than in the control group of islets cultured in FCS. CONCLUSIONS This preliminary series of controlled experiments suggest that coculture of freshly isolated canine islets in a tissue culture medium supplemented with solubilized subintesti- nal submucosa can provide an extracellular matrix and possibly biotrophic factors that can improve postculture recovery and in vitro islet function. Future investigations will focus on the cellular interactions of SIS, both in vitro and in vivo. REFERENCES 1. Badylak SF, Lantz GC, Coffey A, et al: J Surg Res 47:74, 1989 2. Lakey JRT, Cavanagh TJ, Zieger MAJ, et al: Cell Transplant (submitted) 3. Ricordi C, Lacy PE, Finke EH, et al: Diabetes 37:413, 1988 4. Ricordi C, Gray DWR, Hering BJ, et al: Acta Diabetol Lat 27:185, 1988 From the Surgical Medical Research Institute, Department of Surgery, University of Alberta, Edmonton, Canada; Cryobiology Research Institute, Indianapolis, Indiana. Supported by the Cryobiology Research Institute, Indianapo- lis, Ind, and Cook Biotechnology, Purdue University, West Lafay- ette, Ind. J.R.T.L. is supported by the Alberta Heritage Founda- tion for Medical Research. Address reprint requests to J.R.T. Lakey, Surgical Medical Research Institute, University of Alberta, Rm 1074, Dent/Pharm Bldg, Edmonton, Canada T6H 2N8. © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(97)01320-1 Transplantation Proceedings, 30, 383 (1998) 383