Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.
1. Purification of islets
2. Sampling and culture
In spite of significant advancements in techniques of human islet isolation, islet yield remain highly variable and unpredictable. Purification of digested pancreatic tissue is crucial to recover a sufficient isle mass after successful enzymatic digestion for transplantation. The UIC purification method is recommended because a superior recovery of highly pure human islets was demonstrated by using this method. Moreover, up to 50 ml of digested tissue can be loaded in a single Cobe run for purification, thus minimizing the ischemia-associated injury of the human islets by shortening the total time used on isolation.
Supported by founding from the University of Illinois at Chicago as well as Islet Cell Resource Center NIH grant (RFA-RR 05-003), the Christopher Foundation, the Efroymson Foundation, and the Tellabs Foundation.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Centrifuge | equipment | Beckman Coulter | 424803 | |
Cobe 2991 Cell Processor | equipment | Gambro. BCT | 2556 | islet purification |
MZ6 Inverted Microscopy TLBD4.1 | equipment | Leica Diagnostic Instrument Co | 4103 | |
Cell Culture Dish with 2 mm Grid | equipment | Nunc Brand Products | 174926 | islet counting |
Digital Sight DS L1 | equipment | Nikon | 217267 | |
COBE bag | disposable equipment | O.R. Solution | 66707003 | |
T175 culture flask | disposable equipment | Sarstedt | 83.1812.500 | |
University of Wisconcin (UW) solution | reagent | DURAMED | 1000-46-06 | |
Hank’s Buffered Salt Solution (HBSS) | reagent | Mediatech Inc | 99-597-CM | |
Ficoll 1.100 | reagent | Bichrom AG | L6155 | |
M199 media (wash solution) | reagent | Mediatech Inc | 99-784-CM | |
Final wash/Culture Medium | reagent | Mediatech Inc | 99-785-CV | |
Dithizone | reagent | Sigma | D5130 |