Optimal method for short-term or long-term islet preservation: comparison of islet culture, cold preservation and cryopreservation

Abstract

Islet preservation plays an important role for the success of islet transplantation. To determine the optimal method for islet preservation, we compared the outcomes of islet culture, cold preservation, and cryopreservation in this study. Isolated rat islets were divided into three groups: 37 °C group (conventional culture at 37 °C in RPMI-1640 medium), 4 °C group (cold preservation at 4 °C in University of Wisconsin (UW) solution), and -80 °C group (cryopreservation at -80 °C with dimethyl sulfoxide (DMSO)). Recovery rate, Calcein-AM/PI double staining, insulin release, mRNA level of hypoxia-inducible factor-1α (HIF-1α), and protein level of Bax in islets were examined after short-term (1 day) or long-term (7 days) preservation. After either short-term or long-term preservation, 4 °C group showed higher recovery rate of the islets number, lower percentage of PI positive area, better insulin release ability, and lower expression levels of HIF-1α and Bax in comparison to the 37 or -80 °C group. Meanwhile, islets in 37 °C group showed better function, and down-regulation of HIF-1α and Bax than those in -80 °C group on day 1; however, worse function of islets, up-regulated HIF-1α and Bax in 37 °C group were observed in comparison to -80 °C group on day 7. These results suggest that cold preservation at 4 °C in UW solution is the optimal method in comparison to the conventional culture at 37 °C or cryopreservation at -80 °C for short-term or long-term islet preservation. Furthermore, the potential mechanism may relate to, at least in part, apoptosis induced by the HIF-1α.

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“…Calcein-AM/PI double staining was performed as described previously [ 19 ] to determine the cells survival. Calcein AM positive area rate was degined as the percentage of the calcein AM positive area to the whole cells area.…”

“…Western blot analysis was performed as described previously [ 19 ]. In brief, Protein concentration was measured using BCA protein assay Kit (Thermo, USA).…”

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“…Western blot was done as previously described [39]. In brief, cells were lysed and incubated for 30 min in RIPA buffer.…”

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“…Liu et al [41] compared outcomes of conventional islet culture at 37°C in RPMI-1640 medium, cold preservation at 4°C in University of Wisconsin (UW) solution, and cryopreservation at -80°C with dimethyl sulfoxide to determine the optimal method for islet preservation, which is crucial to achieve successful and efficient islet transplantation. After short-term (1 day) or long-term (7 days) preservation, cold preservation at 4°C showed higher recovery rate of the islet number, lower percentage of dead cells to viable cells, and better insulin release ability in comparison with the islet culture at 37°C or cryopreservation at -80°C, suggesting that cold preservation at 4°C in UW solution is the optimal method in comparison with the conventional culture or cryopreservation for short-term and long-term islet preservation.…”