Human Islet Separation Utilizing a Closed Automated Purification System

Abstract

A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 +/- 1.1% vs. 98.2 +/- 2.0%, p < 0.05). Islet yield (120,468 +/- 15,970 vs. 114,570 +/- 15,313 IE, NS) and purity (51.7 +/- 4.8% vs. 54.4 +/- 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.

Glycemic Stability Through Islet-After-Kidney Transplantation Using an Alemtuzumab-Based Induction Regimen and Long-Term Triple-Maintenance Immunosuppression

“…The islet isolation procedure was performed in the Good Manufacturing Practice (GMP) facility at the LUMC using standard operating procedures described earlier (4,17,18). In short, after receiving the donor pancreas in the GMP facility, the organ was prepared and the pancreatic duct was cannulated with a single cannula, after which the organ was perfused with collagenase NB1 and neutral protease (Serva Electropheresis, Germany), aiming for pancreas distension with minimal enzyme leakage.…”

“…In short, after receiving the donor pancreas in the GMP facility, the organ was prepared and the pancreatic duct was cannulated with a single cannula, after which the organ was perfused with collagenase NB1 and neutral protease (Serva Electropheresis, Germany), aiming for pancreas distension with minimal enzyme leakage. The pancreas tissue was digested in a closed loop, collected, and washed, after which the islets were purified using density gradient separation in a COBE 2991 Cell separator (TerumoBCT, Lakewood, CO) (17,18). Finally, the islets were cultured in CMRL-1066 solution (Corning-Mediatech, Manassas, VA) supplemented with 10% ABO-compatible human serum (Sanquin Bloodbank, The Netherlands) until they were transplanted (18).…”

“…The pancreas tissue was digested in a closed loop, collected, and washed, after which the islets were purified using density gradient separation in a COBE 2991 Cell separator (TerumoBCT, Lakewood, CO) (17,18). Finally, the islets were cultured in CMRL-1066 solution (Corning-Mediatech, Manassas, VA) supplemented with 10% ABO-compatible human serum (Sanquin Bloodbank, The Netherlands) until they were transplanted (18). Islet equivalent (IEQ) was calculated after assessment of purity and pellet volume of the islet product (19).…”

Using HTK for Prolonged Pancreas Preservation Prior to Human Islet Isolation

“…Isolation, purification, and culture of human islets were performed as previously described utilizing collagenase NB1 and neutral protease (Serva, Heidelberg, Germany) for pancreas digestion performed at 37 C in a continuous digestion-filtration device [16,19]. Enzyme activity and perfusion volume were adjusted according to the trimmed pancreas weight and administered by means of continuous ductal perfusion as previously reported [20].…”

“…Enzyme activity and perfusion volume were adjusted according to the trimmed pancreas weight and administered by means of continuous ductal perfusion as previously reported [20]. Islet purification was performed in an automated closed purification system utilizing a continuous hyperosmolaric Ficoll gradient for islet separation from non-endocrine tissue [19,21]. Purified islets were resuspended in CMRL 1066 supplemented with 10% ABO-compatible human serum, and cultured for 2 to 3 d in a semi-closed culture bag system (Baxter Medical AB, Stockholm, Sweden) that was incubated in humidified atmosphere (5% CO 2 ) for 1 day at 37 C and subsequently at 24 C [16].…”