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Presenter: Morgan, Lamb, Orange, United States
Authors: Jonathan RT Lakey1, Morgan Lamb1, Andrew Breite2, David Chapman1, Kelly Laugenour1, Ouwen Liang1, Robert McCarthy2
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Comparison of enzyme efficiency in porcine islet isolation and in vitro maturation from young pigs
Jonathan RT Lakey1, Morgan Lamb1, Andrew Breite2, David Chapman1, Kelly Laugenour1, Ouwen Liang1, Robert McCarthy2
1Surgery, University Of California Irvine, Orange, CA; 2VitaCyte LLC, Indianapolis, IN; United States
Islet xenotransplantation continues to be a potential option for treating patients with Type 1 diabetes. Recent FDA industry guidelines encourage tight control of the reagents and methods used in the islet manufacturing process. The aim of this study was to compare islet isolation and maturation of isolated porcine islets using either crude collagenase or a purified collagenase-protease mixture. The enzyme dosages were matched using values provided on the Certificates of Analysis.
Twelve porcine islet isolations were performed using pancreases recovered from young Yorkshire pigs (6-8 kg). Tissue was enzymatically digested with purified Collagenase HA and BP Protease (Group 1) or Type V collagenase (Group 2). The islets were then cultured for 10 days at 37ºC. Isolated islets were assessed for yield, islet equivalents (IE), cell viability using FDA/PI, and function using a glucose stimulated insulin release (GSIR) assay and calculation of stimulation index (SI, ratio of insulin produced in high (28mM) over low glucose (2.8mM)).
Results from these isolations are presented in Table 1, with the results presented at mean ± standard error of the mean (SEM). No significant difference in results between groups.
These studies exhibit that a stable well-defined enzyme product can effectively isolate viable porcine islets, which can be used in clinical islet transplant. Moreover, since the enzyme activity of the purified material is defined, further modifications to the composition can be made to assess the tolerance of islet yield and function to variation in enzyme composition.
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