Survival and Maturation of Microencapsulated Porcine Neonatal Pancreatic Cell Clusters Transplanted into Immunocompetent Diabetic Mice
Differentiation and maturation of porcine neonatal pancreatic cell clusters (NPCCs) microencapsulated in barium alginate were assessed after transplantation into immunocompetent mice. Microencapsulated NPCCs were transplanted into the peritoneal cavity of streptozocin-induced diabetic B6AF1 mice (n ؍ 32). The microcapsules were removed at 2, 6, and 20 weeks and examined for cellular overgrowth, insulin content, and insulin secretory responses to glucose and glucose with theophylline. The
... ophylline. The differentiation, maturation, and proliferation of the ␤-cells in the NPCCs were assessed by immunohistochemistry. Blood glucose levels were normalized in 81% of the animals that received a transplant and remained normal until termination of the experiments at 20 weeks. Hyperglycemic blood glucose levels after explantation of the capsules confirmed the function of the encapsulated NPCCs. Insulin content of the encapsulated NPCCs was increased 10-fold at 20 weeks after transplantation compared with pretransplantation levels. A 3.2-fold increase of the ratio of the ␤-cell area to the total cellular area was observed at 20 weeks, demonstrating the maturation of NPCCs into ␤-cells. In conclusion, NPCCs encapsulated with simple barium alginate can differentiate into ␤-cells and reverse high blood glucose levels in immunocompetent mice without immunosuppression for >20 weeks. Diabetes 52:69 -75, 2003 RESEARCH DESIGN AND METHODS Preparation of NPCCs. NPCCs were prepared using a modification (11) of a technique described by Korbutt et al. (10). Male Yorkshire pigs (1-3 days old; Parsons Farm, Hadley, MA) were anesthetized with 0.15 ml of Telazol (tiletamine HCL; Fort Dodge Laboratories, Ft. Dodge, IA) and 0.30 ml of Xylaject (xylazine HCL; Phoenix Pharmaceutical St. Joseph, MO) given by intramuscular injection. After partial exsanguination, the pancreas was removed through a midline incision. Pancreata were then minced into 1-to 2-mm 3 pieces in a 50-ml plastic Falcon centrifuge tube containing 25 ml of M199 medium. After centrifugation for 1 min at 600 rpm, the supernatant was removed and 5 mg/ml collagenase (collagenase P; Roche, Indianapolis, IN) was added to the pellet. The pancreata were digested in a water bath at 37°C. After filtration, NPCCs were washed two times and were cultured in Ham's F10 media (Life Technologies, Grand Island, NY) supplemented with 10 mmol/l glucose, 10 mol/l 3-isobutyl-l-methylxanthine (Sigma, St. Louis, MO), 2 mmol/l L-glutamine (Life Technologies), 10 mmol/l nicotinamide (Sigma), 25 mg/l CaCl 2 :H 2 O (Sigma), 0.5% BSA (RIA grade Fraction V, Sigma), and antibiotics (100 units/ml penicillin, 100 g/ml streptomycin; Life Technologies) in 150 ϫ 50 mm bacteriological plates (Becton Dickinson, Franklin Lakes, NJ) at 37°C for 8 days. Media changes were performed on days 2, 4, 5, and 7. Microencapsulation. Encapsulation was performed with a minor modification of a previously described technique (15), using a highly purified alginate containing 61% mannuronic acid and 39% guluronic acid (High M alginate 3.3%, the gift of John Holahan, Pharmacia, Peapack, NJ). At day 8 after preparation, NPCCs were washed with M199 medium and calcium-free Krebs Ringer buffer.