Comparison of Size, Viability, and Function of Fetal Pig Islet-Like Cell Clusters after Digestion Using Collagenase or Liberase

Abstract

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 µm) and Liberase (79 ± 0.4 µm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

“…We also suspect that Liberase has a gentler action than did standard crude collagenase preparations and may require a longer time to act. Indeed, other authors have reported longer digestion times for other tissues such as pancreas (35) and ovary (36) when using Liberase compared with standard collagenase.…”

Efficient Isolation of Cardiac Stem Cells from Brown Adipose

“…Different protease activity was necessary for optimal tissue digest efficacy and cell yield [19, 24–26]. However, the challenge is to balance the combination of enzymes and digesting time for the desired cell yield and function [27].…”

Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies

“…The method for extraction and purification of islets from the pig pancreas has undergone significant improvements and has been reported for fetal [15–17], neonatal [18,19], and adult pigs [20–23]. Insulin‐producing beta cells in fetal and neonatal pig pancreata are organized in cell clusters that lack a defined shape, unlike adult islets, and therefore are very difficult to separate from the exocrine pancreatic cells and other non‐islet cells of the pancreas.…”