Advantages of Using a Cell Separator and Metrizamide Gradients for Human Islet Purification1

Abstract

Human islet transplantation has a high rate of failure, often due to primary nonfunction, which suggests that islets are damaged during the processing of the pancreas. The preparation of human islets for transplantation is still a complex process that requires large teams of surgical and laboratory personnel. To overcome this problem, we have adopted the use of the IBM 2991 COBE cell separator and a metrizamide/Ficoll density medium that is easy to prepare. Twenty-seven pancreatic glands have been processed using the COBE cell separator, 23 of which were purified in metrizamide/Ficoll gradients and 4 in bovine serum albumin gradients. The results show an improvement of recovery and viability in these preparations when compared retrospectively with manual gradients. More importantly, the time required for purification was shortened to one fourth the usual time and total processing time is about half as long. Moreover, a team of two laboratory staff was regularly able to prepare islets for transplantation, reducing the separation time from 7 hr to 3.5 hr. We conclude that the automatic cell separator and metrizamide-based separation medium are useful modifications of current islet purification methods.

A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets

“…In β pancreatic and rat insulinoma INS-1E cells, where Ca 2 + concentration is around 100 nM already at resting condition (2.5 mM glucose) [28], TG2 mainly functions as a transglutaminase, increasing its transamidating activity during first phase insulin secretion [25] when Ca 2 + concentration reaches the value of 450 nM in INS-1E [28] and 300 nM in isolated islets [41]. In the model we previously proposed [27], TG2 during first phase insulin secretion acts as a modifying enzyme by inserting post translational modification(s) in various proteins both in the cytoplasm and in the granular/microsomal fraction, [25,42].…”

“…Islets were isolated according to the automated method [26], purified by continuous gradient with refrigerated COBE processor as previously described [27] and maintained in M199 medium, supplemented with serum and antibiotics, at 37 °C, 5% CO 2 until use. M199 was chosen because its glucose concentration is 5.5 mM.…”

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

“…Two types of enzymes were used: collagenase type P (1-3 mg/ml) and liberase (0.5-1.4 mg/ml) (Roche, Indianapolis, IN, USA). Islets were purified by discontinuous gradient in syringes (density gradient: 1,108; 1,096; 1,037: Euroficoll, Sigma-Aldrich, Milan, Italy), or by continuous gradient with refrigerated COBE processor as previously described [10]. After isolation, islets were cultured at 22°C in a humidified atmosphere (5% CO 2 ), in M199 medium (Euroclone, Celbio, Milan, Italy) or CMRL (Mediatech, Cellgro, VA, USA) supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin sulphate (Euroclone, Celbio) and 2 mmol/l glutamine (Mediatech, Cellgro, VA, USA).…”

Characterization of Collagenase Blend Enzymes for Human Islet Transplantation

“…Islets were isolated using the automated method described by Ricordi (23) and purified as previously described (24). The enzyme used for isolation was Liberase™ (0.5-1.4 mg/mL; Roche, Indianapolis, IN).…”