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 I: digestion and collection of pancreatic tissue) using a modified automated method.
1. Facility setup
2. Pancreas preparation and perfusion
3. Pancreas digestion
4. Tissue collection
There are a variety of factors affecting the isolation outcome 9,10. Among them, digestion of the pancreatic tissue plays a prominent role. Based on our own human islet isolation experience, we summarized following critical points, which could potentially influence the islet yield and quality.
Supported by funding 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 |
---|---|---|---|---|
Ice Slush Machine | equipment | Taylor Company | K4036546 | make sterile ice |
Centrifuge | equipment | Beckman Coulter | 424803 | |
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 Water Bath EX7 | equipment | NesLab | 105361058 | |
Thermometer | equipment | IBI:IR | 60444 | |
MonoBloc Balance AB 104-S | equipment | Metler Toledo | 1119430864 | measure pancreas weight |
MasterFlex II Speed Controller | equipment | Cole Parmer Instrument Co | J00006445 | adjust flow speed during degestion |
Digital Sight DS L1 | equipment | Nikon | 217267 | |
Perfusion Unit | equipment | Custom Made/Swiss | n/a | |
Cooler for perfusion unit | equipment | Fisher Scientific | 3013P | |
Ricordi Chamber | equipment | University of Miami | n/a | |
RPMI dilution solution | reagent | Mediatech Inc | 99783024 | |
University of Wisconcin (UW) solution | reagent | DURAMED | 1000-46-06 | |
Hank’s Buffered Salt Solution (HBSS) | reagent | Mediatech Inc | 99-597-CM | |
25% Human Albumin | reagent | GRIFOLS | NDC 61953-0002-2 | |
M199 media (wash solution) | reagent | Mediatech Inc | 99-784-CM | |
Collagenase NB1 GMP grade | reagent | SERVA Electrophoresis | N0002937 | |
Neutral Protease NB1 GMP grade | reagent | SERVA Electrophoresis | N0002936 | |
Betadine | reagent | Cardinal Health | 29906-004 | |
Cefazolin | reagent | West-Ward Pharmaceutical | NDC 0143-9924-90 | |
Dithizone | reagent | Sigma | D5130 |