Improved hypothermic short-term storage of isolated mouse islets by adding serum to preservation solutions

Abstract

Preserving isolated islets at low temperature appears attractive because it can keep islet quantity comparable to freshly isolated islets. In this study, we evaluated the effect of serum as an additive to preservation solutions on islet quality after short-term hypothermic storage. Isolated mouse islets were preserved at 4°C in University of Wisconsin solution (UW) alone, UW with serum, M-Kyoto solution (MK) alone or MK with serum. We then assessed islet quantity, morphology, viability and function in vitro as well as in vivo. Islet quantity after storage in all four solutions was well maintained for up to 120 h. However, islets functioned for different duration; glucose-stimulated insulin release assay revealed that the duration was 72 h when islets were stored in UW with serum and MK with serum, but only 24 h in UW alone, and the islet function disappeared immediately in MK alone. Viability assay confirmed that more than 70% islet cells survived for up to 48 h when islets are preserved in UW with serum and MK with serum, but the viability decreased rapidly in UW alone and MK alone. In in vivo bioassays using 48-h preserved isogeneic islets, all recipient mice restored normal blood glucose concentrations by transplants preserved in UW with serum or MK with serum, whereas 33.3% recipients and no recipient restored diabetes by transplants preserved in UW alone and in MK alone respectively. Adding serum to both UW and MK improves their capability to store isolated islets by maintaining islet functional viability.

Keywords: M-Kyoto solution; University of Wisconsin solution; hypothermic short-term storage; islet; serum.

Figure 1. Islet quality, total volume and morphology during hypothermic storage in preservation solutions with or without serum. Isolated mouse islets were stored at 4°C in UW alone (open circle), UW with serum (closed circle), MK alone (open triangle) or MK with serum (closed triangle). (A) Number of islets for 120 h in preservation solutions. (B) Total volume of islets for 120 h in preservation solutions. (C–F) Representative histopathological images of the islets stored for 48 h in each group. (G) Representative histopathological image of the islets freshly isolated. Scale bar = 50μm

Figure 2. Glucose-stimulated insulin release of preserved islets. Preserved islets of each experimental group were subjected to static glucose challenge. One experiment contains two sequential sets of challenge in which 3.3 mmol/L of low glucose concentration (L) and 20 mmol/L of high glucose concentration (H) were used in succession.

Figure 3. Viability assay by analyzing islet membrane integrity. Preserved islets stained with fluorescent dye were evaluated using confocal laser scanning microscope. (A–D) Representative photo images of islets preserved for 48 h in UW alone (A), UW with serum (B), MK alone (C) and MK with serum (D). Each figure has four images; bright field (upper left), YOYO-1 positive cells (upper right), Hoechst 33342 positive cells (lower left), and a merged image of YOYO-1 positive with Hoechst 33342 (lower right). (E) Time course change of viability of preserved islets.

Figure 4. In vivo function of preserved islets. (A–C) Changes in the blood glucose concentration of mice after 300 preserved isogeneic islets were transplanted as bioassay. (A) Islets preserved for 48 h in UW alone (open circle) and in UW with serum (closed circle) were used as transplant. (B) Islets preserved for 48 h in MK alone (open triangle) and in MK with serum (closed triangle) were used as transplant. (C) Islets freshly isolated were used as transplant. (D) Intraperitoneal glucose tolerance test 32 d after transplantation.

Figure 5. Histopathological analysis for sections of the resected left kidney bearing transplanted islets in its subrenal capsule. Hematoxylin and eosin (H&E) stain and immunohistochemical anti-insulin stain were performed. Original magnification 100x; Scale bar = 150 μm.