Inhibition of chloride ion influx into islet cells protects islets during pancreatic islet isolation

T. Anazawa¹, T. Tsuchiya¹, A. Kenjo¹, J. Haga¹, T. Sato¹, N. Sato¹, T. Saito¹, Y. Sato¹, M. Miyake², A. Hazama², M. Gotoh^1
1 Fukushima Medical University, Surgery, Fukushima, Japan; ² Fukushima Medical University, Physiology, Fukushima, Japan

Introduction: It is known that the increase of the plasma cell membrane permeability with subsequent chloride ion influx causes cell death. We examined the effects of a chloride channel blocker, 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid disodium salt (DIDS) and extracellular Cl^--free conditions on islet isolation outcomes.

Methods: We monitored islet cells incubated with collagenase and proteolytic enzymes to determine whether collagenase digest induces islet cell death through Cl^- influx into the cells. Experimental groups were created according to the collagenase solutions used for Wistar rat islet isolation: control group, HBSS; DIDS group, 200 µM DIDS was added to HBSS; and Cl^--free group, Cl^--free HBSS was created by replacing sodium chloride by sodium gluconate. We assessed islet yield and viability of isolated islets using both in vitro and in vivo assays.

Results: We observed an increase in the intracellular Cl^- concentration under collagenase digest condition using a Cl^- sensitive fluorescent dye, subsequently the rupture of islet cells. Consequently, the islet yields were significantly higher in the DIDS and Cl^--free groups than in the control group, and the islet membrane integrity of these groups was preserved. Among STZ-induced diabetic SCID mice transplanted under renal subcapsular space with marginal dose islets, all 7 mice from the DIDS or 6 of 7 mice in the Cl^--free groups became normoglycemic, compared to 2 of 7 mice in the control group (control vs DIDS, p=0.010; control vs Cl^--free, p=0.051). The blood glucose curve in the IPGTT for the DIDS group and the Cl^--free group indicated excellent in vivo islet function.

Conclusion: Our results suggested that Cl^- influx into the cells causes the cell damage during the digestion procedure. Inhibition of Cl^- influx into islets by DIDS or Cl^--free condition protects islets, offering a new strategy for improving human islet isolation outcome.

MO-138