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Beta cell mass after transplantation of cryopreserved islets
Beta Cell Mass After Transplantation of Cryopreserved Islets V. Nacher, M. Pe´rez-Maraver, R. Jara, J. Soler, and E. Montanya
T
HE LIMITED NUMBER of donors and the difficulty in isolating a sufficient number of islets from a single human pancreas have been major obstacles to successful islet transplantation. Cryopreservation remains the most practical method for long-term storage of islet tissue. However, previous studies have shown that a larger number of cryopreserved islets is required to reverse diabetes when compared with nonfrozen islets.1–2 Cryopreserved islets could be more susceptible to beta cell destruction after transplantation than noncryopreserved islets and higher beta cell loss could contribute to the requirement for a higher number of cryopreserved islets. The goal of the study was to determine the beta cell mass in cryopreserved and noncryopreserved islets exposed to the same metabolic conditions after syngeneic islet transplantation. MATERIALS AND METHODS Streptozocin-diabetic Lewis rats (blood glucose 28.4 ⫾ 0.7 mmol/L) were transplanted with 1400 syngeneic islets (700 cryopreserved islets under one kidney capsule and 700 noncryopreserved islets under the contralateral kidney capsule). Isolated islets were cultured overnight in RPMI-1640 medium supplemented with 10% fetal calf serum. After this culture period, islets were cultured for an additional 24 hours and transplanted (noncryopreserved transplantation) or cryopreserved as described by Rajotte.3 After thawing, cryopreserved islets were cultured for 24 hours and transplanted (cryopreserved islets). Insulin secretion was determined in static incubations at 5.5 mmol/L and 16.7 mmol/L glucose. The grafts were obtained 4 (group 1), 14 (group 2), or 56 days (group 3) after transplantation. Grafts were weighed and processed for paraffin embedding. Sections were stained by immunoperoxidase with a guinea-pig anti-insulin antibody and beta cell mass was measured by point counting morphometry as previously described.4 The beta cell mass of islets at the time of transplantation was determined in four groups of 700 cryopreserved or 700 noncryopreserved islets, isolated on different days.
RESULTS AND DISCUSSION
After cryopreservation, the mean islet recovery was 85 ⫾ 2%. Insulin secretion was similar in cryopreserved and noncryopreserved islets (5.5 mmol/L glucose: 2.8 ⫾ 1.2 ng 䡠 10 islets⫺1 䡠 60 min⫺1 and 3.1 ⫾ 0.6 ng 䡠 10 islets⫺1 䡠 60 min⫺1; 16.7 mmol/L glucose: 51.8 ⫾ 15.0 ng 䡠 10 islets⫺1 䡠 60
0041-1345/99/$–see front matter PII S0041-1345(99)00500-X 2560
min⫺1 and 32.7 ⫾ 9.6 ng 䡠 10 islets⫺1 䡠 60 min⫺1, in cryopreserved and noncryopreserved islets, respectively). Blood glucose was 12.5 ⫾ 2.0 mmol/L in group 1, 9.7 ⫾ 2.2 mmol/L in group 2, and 5.3 ⫾ 0.3 mmol/L in group 3, when the grafts were harvested (normal: 5.3 ⫾ 0.2 mmol/L). Beta cell mass was similar in grafts from cryopreserved and noncryopreserved islets (group 1: 0.54 ⫾ 0.09 mg and 0.52 ⫾ 0.04 mg; group 2: 1.25 ⫾ 0.39 mg and 1.42 ⫾ 0.32 mg; group 3: 1.23 ⫾ 0.27 mg and 1.42 ⫾ 0.17 mg, respectively) and lower than the initially cryopreserved and noncryopreserved transplanted beta cell mass (2.66 ⫾ 0.17 mg and 2.93 ⫾ 0.23 mg, respectively) (P ⬍ .05). In summary, beta cell mass was similar in grafts of cryopreserved and noncryopreserved islets at any time point after syngeneic transplantation. The similar reduction in beta cell mass in both groups on day 4 after transplantation suggests that beta cell destruction was not higher in cryopreserved islets. On the other hand, the increment in beta cell mass on days 14 and 60 indicates that beta cell growth was similar in cryopreserved and noncryopreserved islets. Therefore, the requirement of a larger number of islets to restore normoglycemia would not be explained by a reduced beta cell mass in cryopreserved transplanted islets. REFERENCES 1. Rajotte RV, Evans MG, Warnock GL, et al: Horm Metab Res 25 (Suppl):72, 1990 2. Rich SJ, Swift S, Thirdborough SM, et al: Transplant Proc 26:823, 1994 3. Warnock GL, Rajotte RV: Cryobiology 26:103, 1989 4. Nacher V, Raurell M, Merino JF, et al: Diabetes 45:1541, 1996
From the Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit, Ciutat Sanita`ria i Universita`ria de Bellvitge (CSUB), Barcelona, Spain. Supported by grant SAF 97-118 (E.M.). Victor Nacher was the recipient of a grant from the Fundacio´ Catalana de Trasplantament. Address reprint requests to Eduard Montanya, Endocrine Unit, Ciutat Sanita´ria i Universita`ria de Bellvitge, Feixa Llarga s/n, 08907-L’Hospitatet de Llobregat, Barcelona, Spain.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 31, 2560 (1999)
T
HE LIMITED NUMBER of donors and the difficulty in isolating a sufficient number of islets from a single human pancreas have been major obstacles to successful islet transplantation. Cryopreservation remains the most practical method for long-term storage of islet tissue. However, previous studies have shown that a larger number of cryopreserved islets is required to reverse diabetes when compared with nonfrozen islets.1–2 Cryopreserved islets could be more susceptible to beta cell destruction after transplantation than noncryopreserved islets and higher beta cell loss could contribute to the requirement for a higher number of cryopreserved islets. The goal of the study was to determine the beta cell mass in cryopreserved and noncryopreserved islets exposed to the same metabolic conditions after syngeneic islet transplantation. MATERIALS AND METHODS Streptozocin-diabetic Lewis rats (blood glucose 28.4 ⫾ 0.7 mmol/L) were transplanted with 1400 syngeneic islets (700 cryopreserved islets under one kidney capsule and 700 noncryopreserved islets under the contralateral kidney capsule). Isolated islets were cultured overnight in RPMI-1640 medium supplemented with 10% fetal calf serum. After this culture period, islets were cultured for an additional 24 hours and transplanted (noncryopreserved transplantation) or cryopreserved as described by Rajotte.3 After thawing, cryopreserved islets were cultured for 24 hours and transplanted (cryopreserved islets). Insulin secretion was determined in static incubations at 5.5 mmol/L and 16.7 mmol/L glucose. The grafts were obtained 4 (group 1), 14 (group 2), or 56 days (group 3) after transplantation. Grafts were weighed and processed for paraffin embedding. Sections were stained by immunoperoxidase with a guinea-pig anti-insulin antibody and beta cell mass was measured by point counting morphometry as previously described.4 The beta cell mass of islets at the time of transplantation was determined in four groups of 700 cryopreserved or 700 noncryopreserved islets, isolated on different days.
RESULTS AND DISCUSSION
After cryopreservation, the mean islet recovery was 85 ⫾ 2%. Insulin secretion was similar in cryopreserved and noncryopreserved islets (5.5 mmol/L glucose: 2.8 ⫾ 1.2 ng 䡠 10 islets⫺1 䡠 60 min⫺1 and 3.1 ⫾ 0.6 ng 䡠 10 islets⫺1 䡠 60 min⫺1; 16.7 mmol/L glucose: 51.8 ⫾ 15.0 ng 䡠 10 islets⫺1 䡠 60
0041-1345/99/$–see front matter PII S0041-1345(99)00500-X 2560
min⫺1 and 32.7 ⫾ 9.6 ng 䡠 10 islets⫺1 䡠 60 min⫺1, in cryopreserved and noncryopreserved islets, respectively). Blood glucose was 12.5 ⫾ 2.0 mmol/L in group 1, 9.7 ⫾ 2.2 mmol/L in group 2, and 5.3 ⫾ 0.3 mmol/L in group 3, when the grafts were harvested (normal: 5.3 ⫾ 0.2 mmol/L). Beta cell mass was similar in grafts from cryopreserved and noncryopreserved islets (group 1: 0.54 ⫾ 0.09 mg and 0.52 ⫾ 0.04 mg; group 2: 1.25 ⫾ 0.39 mg and 1.42 ⫾ 0.32 mg; group 3: 1.23 ⫾ 0.27 mg and 1.42 ⫾ 0.17 mg, respectively) and lower than the initially cryopreserved and noncryopreserved transplanted beta cell mass (2.66 ⫾ 0.17 mg and 2.93 ⫾ 0.23 mg, respectively) (P ⬍ .05). In summary, beta cell mass was similar in grafts of cryopreserved and noncryopreserved islets at any time point after syngeneic transplantation. The similar reduction in beta cell mass in both groups on day 4 after transplantation suggests that beta cell destruction was not higher in cryopreserved islets. On the other hand, the increment in beta cell mass on days 14 and 60 indicates that beta cell growth was similar in cryopreserved and noncryopreserved islets. Therefore, the requirement of a larger number of islets to restore normoglycemia would not be explained by a reduced beta cell mass in cryopreserved transplanted islets. REFERENCES 1. Rajotte RV, Evans MG, Warnock GL, et al: Horm Metab Res 25 (Suppl):72, 1990 2. Rich SJ, Swift S, Thirdborough SM, et al: Transplant Proc 26:823, 1994 3. Warnock GL, Rajotte RV: Cryobiology 26:103, 1989 4. Nacher V, Raurell M, Merino JF, et al: Diabetes 45:1541, 1996
From the Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit, Ciutat Sanita`ria i Universita`ria de Bellvitge (CSUB), Barcelona, Spain. Supported by grant SAF 97-118 (E.M.). Victor Nacher was the recipient of a grant from the Fundacio´ Catalana de Trasplantament. Address reprint requests to Eduard Montanya, Endocrine Unit, Ciutat Sanita´ria i Universita`ria de Bellvitge, Feixa Llarga s/n, 08907-L’Hospitatet de Llobregat, Barcelona, Spain.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 31, 2560 (1999)