Improved method for the isolation and purification of human islets of langerhans using Liberase enzyme blend.
To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of 1Hypaque EuroFicoll. Isolations in Group I (Sevac) had an average yield of 138,602 +/- 128,364 islet equivalents (IE) (2083 +/- 1679 IE/g) with a purity of 85 +/- 11%. Group II (Liberase) showed an average yield of 389,586 +/- 191,161 IE (5,958 +/- 3,083 IE/g) with a purity of 90 +/- 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.
Olack
B J
BJ
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Swanson
C J
CJ
Howard
T K
TK
Mohanakumar
T
T
D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D017364:Collagenases; D005260:Female; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D008297:Male; D008875:Middle Aged; D011933:Reagent Kits, Diagnostic; D013820:Thermolysin
D011933:Reagent Kits, Diagnostic; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin