Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Summary

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.

Abstract

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients ¹, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome ². At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient ³. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) ^4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al ⁸. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification ³. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods ⁹. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37°C overnight culture before islets were transplanted or used for research.

Protocol

1. Purification of islets

1. To start the islet purification procedure, load the COBE bag, clamp all tubes except the green tube, and set the speed to 1,500 and the super out to 0.
2. In the hood set up the pump and gradient beakers and connect the collection tubing to the green tube.
3. Now the loading of the ficoll 1.100 can begin: push “spin” then add 110 ml of Ficoll 1.100 to the front beaker, and start the pump in order to load the COBE bag. As the Ficoll is loaded, press super out, stop the pump, unclamp the pump head and set the super out speed to 100.
4. When the ficoll reaches the beaker and all air is expelled from the ....

Discussion

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 t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge	equipment	Beckman Coulter Inc.	424803
Cobe 2991 Cell Processor	equipment	Gambro. BCT	2556	islet purification
MZ6 Inverted Microscopy TLBD4.1	equipment	Leica Microsystems	4103
Cell Culture Dish with 2 mm Grid	equipment	Nalge Nunc international	174926	islet counting
Digital Sight DS L1	equipment	Nikon Instruments	217267
COBE bag	disposable equipment	O.R. Solution	66707003
T175 culture flask	disposable equipment	Sarstedt Ltd	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-Aldrich	D5130