Isolation and preservation of islets from the mouse, rat, guinea-pig and human pancreas

Abstract

Viable islets of Langerhans have been isolated from the mouse, rat, guinea-pig and human pancreas using a free hand microdissection procedure. Viablity has been assessed by light microscopy of sections stained with Gomori's aldehyde fuchsin and by measuring the insulin release from islets in vitro in response to a glucose stimulus. Ten pieces of human cadaver pancreas have been studied. Islets were isolated from 6 and in 5 cases were shown to respond to a glucose stimulus in vitro. Five pieces of human pancreas removed at operation have been studied. Islets were isolated in all cases but only 2 showed a response to a glucose stimulus. Isolated animal islets have been subjected to three preservation systems and their viability following storage noted. 1. Simple cold storage in Hank's balanced salt solution at 4 degrees C. At 15 hours 100% survival was noted. This dropped to 10% at 48 hours. There were no survivors at 72 hours. 2. Subzero storage. In group I (freezing rate 1 degree C/min) histological survival was 35% and functional survival 20%. In group II (freezing rate 5 degrees C/min with a 24-hour culture period after rewarming) histological survival was approximately 87% and functional survival 75%. 3. Organ culture. Islets from the guinea-pig, rat and mouse showed minimal morphological damage when cultured for 21 days in a simple organ culture system. At 28 days histological survival was approximately 30%. We were unable to correlate histological and functional survival in this group.

Cryogenic preservation of isolated rat Islets of Langerhans: Effect of cooling and warming rates

“…in the presence of DMSO and fetal calf serum and at least 50% of the islets "survived" based on histological criteria. A subsequent repor~ by Ferguson et al [9] extended these observations to islets isolated from mice, guinea pigs and humans. They report no histological or functional survival for mouse islets and a highly variable response for human islets frozen at 5~ Rajotte et al suspended rat Islets of Langerhans in either 7.5 or 10% V/V DMSO and frozen at approximately 0.6~ to -100 ~ followed by rapid warming in a 37 ~ water bath [18].…”

“…The development of techniques for long-term storage without loss of either morphological integrity or physiological capacity, would simplify many types of long term studies on the biochemical and phy-siological properties of Islets of Langerhans and will be a fundamental step in the development of a practical method for transplantation of islets, between noninbred individuals. Several previous attempts have been made to use low-temperature techniques for adult islet preservation [8,9,11]; however, quantitative estimates of survival from these studies were not reported. Here we report the initial results of a survey of the major variables in the cryogenic preservation of rat Islets of Langerhans.…”

A Practical Guide to Rodent Islet Isolation and Assessment

“…Although these two approaches form the foundation for many islet isolation techniques, there are considerable variations in the details among published methods, as well as alternative methods for isolating islets [7-11]. The advantages of the CBD method described by Gotoh et al are twofold: (1) perfusing the pancreas through the CBD allows collagenase to access the islets using anatomical structures, and (2) stationary digestion reduces mechanical damage to the islet.…”

Islet Cryopreservation Protocols

“…25 Over the years many studies have reported optimum cryopreservation of islets; however, comparison of various freezing protocols has been difficult because of the variation in the methods used for freezing and thawing. When our slow cooling protocol was compared with that of others that have been cited as optimum, 13,14,[16][17][18][19][20][21][22][23][24][25][26] we found that slow cooling to −40°C in combination with rapid thawing from −196°C gave the best results as assessed by perifusion and transplantation. 27,28 This has been confirmed by another group who, when comparing various protocols, found that slow cooling to −40°C and rapid thawing from −196°C resulted in the best in vivo survival following the freeze-thaw process.…”