Rat Pancreatic Islet Size Standardization by the “Hanging Drop” Technique

Abstract

Introduction Rejection and hypoxia are the main factors that limit islet engraftment in the recipient liver in the immediate posttransplant period. Recently authors have reported a negative relationship of graft function and islet size, concluding that small islets are superior to large islets. Islets can be dissociated into single cells and reaggregated into so called “pseudoislets,” which are functionally equivalent to intact islets but exhibit reduced immunogenicity. The aim of our study was develop a technique that enabled one to obtain pseudoislets of defined, preferably small, dimensions. Materials and Methods Islets were harvested from Lewis rats by the collagenase digestion procedure. After purification, the isolated islets were dissociated into single cells by trypsin digestion. Fractions with different cell numbers were seeded into single drops onto cell culture dishes, which were inverted and incubated for 5 to 8 days under cell culture conditions. Newly formed pseudoislets were analyzed for dimension, morphology, and cellular composition. Results The volume of reaggregated pseudoislets strongly correlated with the cell number ( r 2 = .995). The average diameter of a 250-cell aggregate was 95 ± 8 μm (mean ± SD) compared with 122 ± 46 μm of freshly isolated islets. Islet cell loss may be minimized by performing reaggregation in the presence of medium glucose (11 mmol/L) and the GLP-1 analogue Exendin-4. Morphology, cellular composition, and architecture of reaggregated islets were comparable to intact islets. Conclusion The “hanging drop” culture method allowed us to obtain pseudoislets of standardized size and regular shape, which did not differ from intact islets in terms of cellular composition or architecture. Further investigations are required to minimize cell loss and test in vivo function of transplanted pseudoislets.