Secretion from Islets and Single Islet Cells following Cryopreservation

Abstract

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen–thawed canine islets and β-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22°C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to −40°C before storage in liquid nitrogen (−196°C). Frozen islets were rapidly thawed (200°C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 ± 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen–thawed islets with a calculated stimulation index of 10.4 ± 1.5 (mean ± SEM) for the frozen–thawed islets, compared with 12.4 ± 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single β-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single β-cells in real time. Secretion from single β-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen–thawed β-cells with an average of 7.0 ± 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 ± 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen–thawed islets. In addition, β-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that β-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.