Experimental Islet Cell Transplantation in Rats: Optimization of the Transplantation Site A. Refaie, M. Gabr, I. Mahmoud, M.A. Bakr, M. El-Baz, and M.A. Ghoneim T HE optimal transplantation site for pancreatic islets continues to be an important issue. 1 The testis has been shown to be an immune privileged site for transplan- tation of various tissues including islets. 2 Some reports have shown that the survival and function of islet allografts is achieved only after this organ had been placed in the abdominal cavity. 3,4 The present three-phases prospective randomized study evaluated this immune privileged site in comparison with other sites, namely the kidney capsule and the liver. MATERIALS AND METHODS Animals Female Sprague-Dawley (SD) rats weighing 150 to 200 g were used as islet donors while male SD rats weighing 200 to 250 g were used as islet recipients. Animals were fed a standard chow pellet diet and tap water and were not fasted prior to experimentation. Rats were made diabetic via single intravenous (IV) injection of streptozoto- cin (Sigma Chemicals Co, St Louis, Mo) at a dosage of 50 mg/kg body weight. The diabetic animals were kept in metabolic cages for daily assessment of body weight, 24-hour urine volume, and urinary glucose level, while random blood sugar and insulin levels were examined weekly. Only rats with persistent blood glucose levels higher than 350 mg/dL for more than 1 week were transplanted. The rat was considered cured if random blood sugar level was below 200 mg/dL, with no glucosuria and normal 24-hour urine volume with steady weight gain. Study Design In phase A, 40 diabetic rats were randomly divided into two groups. The first group received islets in the scrotal testes while in the other group the islets were grafted in the intra-abdominal testes. In phase B, three organ sites were studied: the liver via the portal vein, the renal subcapsular space, and the intra-abdominal testis. Twenty rats were transplanted in each site. In phase C, 60 rats were divided randomly into three groups, islets under the renal subcapsular space in one group and in combination with Sertoli cells in another group. Twenty rats transplanted in the intra-abdominally placed testes served as the control group. Islet Isolation and Culture Islets were isolated in the first two phases according to the method of James et al 5 using collagenase P (Boehringer Mannheim, Indianapolis, Ind). The method of London et al 6 was applied for islet isolation in the third phase of the study using collagenase type XI (Sigma Chemical Co, St Louis, Mo) in order to improve both the yield and quality of the isolated islets. The islets were cultured for 3 to 4 days at 37°C in a humidified atmosphere in the presence of 5% CO 2 . Sertoli Cell Isolation Sertoli cells were isolated according to the method described by Cheng and Bardin 7 using the testes of SD rats 16 to 20 days old. The isolated cells were freshly transplanted under the renal cap- sules with the islet grafts. Each rat received the equivalent of the total amount of Sertoli cells contained in a single testis. Islet Transplantation. Each rat received 10 islets per gram body weight without adjuvant immunosuppression. The islets were injected into the testis through a 26-gauge needle. For the intra-abdominally translocated testis, the grafted gonad was anchored into the abdominal cavity via suturing the gubernaculum to the abdominal muscles. The left kidney was explored and the islets were injected under the renal capsule using a 27-gauge needle without making an incision into the capsule. In case of the combined islets and Sertoli cells, Sertoli cells were injected first, producing a milkish bubble underneath the capsule. The islets were then immediately injected into the bubble. From the Urology and Nephrology Center, Mansoura Univer- sity, Mansoura, Egypt. Address reprint requests to Prof Mohamed A. Ghoneim, MD, MD (HON), Prof of Urology, Director, Urology and Nephrology Center, Mansoura Egypt. Table 1. Homogenicity of the Three Studied Groups Before Transplantation (Phase II) Testis Liver Kidney P Body weight (g) 182.3  22.8 183.2  31.2 181.4  27.2 .98 Duration of diabetes (d) 12.2  8.3 14.3  6.9 13.8  6.8 .66 Random blood sugar (mg/dL) 447  34.5 451.8  57.3 495.5  58.1 .74 Serum Insulin (U/mL) 3.2  2.4 2.3  1.9 2  1.9 .25 0041-1345/98/$19.00 © 1998 by Elsevier Science Inc. PII S0041-1345(97)01328-6 655 Avenue of the Americas, New York, NY 10010 400 Transplantation Proceedings, 30, 400–403 (1998)