A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Summary

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse.

Abstract

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: α cells secrete glucagon, β cells secrete insulin, δ cells secrete somatostatin, ε cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are β cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250—350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

Introduction

There are two common methods in the literature for pancreatic islet isolation. One requires excising the pancreas and dicing it into small pieces using surgical scissors, and then digesting it in a collagenase solution^1,2,3. Another more precise method is to use the network of ducts present in the pancreas to introduce digestive enzyme. The following sites have been used for digestive enzyme injection: the junction of the bile and cystic duct, the gallbladder into the common bile duct, or the common bile duct itself^1,4

Protocol

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Texas A&M University. The surgical tools need is shown in Figure 1 and the schematic diagram of the procedure is shown in Figure 2.

1. Solutions

1. Prepare Hank’s Balanced Salt Solution (HBSS) by adding 100 mL of 10X HBSS (from stock) to 900 mL of distilled water to make 1 L HBSS (1X).
2. Prepare STOP solution (must be made fresh and should be used within 1 h) by adding 50 mL of 100X fetal bovine serum (FBS) to 450 mL of ice cold 1X HBSS; this makes 500 mL STOP sol....

Results

Proper completion of this procedure requires some understanding of mouse anatomy in the abdominal cavity. This allows for proper identification of the ampulla of Vater and clamping of the common bile duct. The entire procedure normally takes 1–2 h. It is more efficient to isolate islets from 4–6 mice at the same time, so several samples can be centrifuged together. The time for islet-picking varies, depending on the number of islets and the efficiency of digestion; it may take roughly an hour to pick 250–350 islets from .......

Discussion

This protocol includes collagenase perfusion and digestion, followed by purification of islets. The most critical steps of this protocol are effective injection and complete perfusion of the pancreas^1,4,7. The delivery method of this protocol allows the enzyme to traverse the anatomical routes to better digest the exocrine tissue surrounding the islets¹. In addition, this technique is well suited for comp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3 mL syringe	BD	309657	Hoding collagenase P
Coverglass forceps	VWR	82027-396	Holding skin of mouse to aid incision procedure
Curved forceps	Sigma-Aldrich	Z168696	Holding tissues during pancreas removal
Isoflurane	Piramal	B13B16A	To anaesthetize mice prior surgery
100 mm petri dishes	VWR	30-2041	Used for islet culture
30 G. ½ inch needle	BD	305106	For penetration of Ampulla of vater to deliver Collagenase P - this guage is used as it fits well in most CBDs
50ml tube	VWR	89039-658	Holding digested pancreatic tissue, collagenase P, and purified islets
Absorbent pads with waterproof moisture barrier	VWR	82020-845	To absorb blood from syurgical procesdudes
Centrifuge 5810R with swing bucket and deceleration capability	Eppendorf	5811FJ478114	Use for pelleting tissues, pellet is formed at bottom of conical tube - swing bucket centrifuge is needed. Also the decelaration feature is important to form the gradient layers.
Collagenase P- 1g	Roche Diagnostics	11249002001	For digestion of exocrine pancreas
Curved surgical scissors	Fisher-Scientific	13-804-21	For cutting open mouse abdomen
Dissection microscope	Olympus	SZX16	Used for identification of key anatomical structures to accurately deliver collagenase into pancreas
Hank's Balanced Salt Solution 10x	Corning	20-023-CV	Washing cells
Histopaque-1077	Sigma	RNBF5100	For gradient formation
Light source	Leeds	LR92240	Enhancing visibility of microscope
RNaseZap	Fisher-Scientific	AM9780	For removing RNase
RPMI-1640 Media w/o L-Glutamine	Corning	15-040-CV	Culturing Islets
Schwartz micro serrefines (Microvascular clamp)	Fine Science Tools	18052-01	Clamping common bile duct and hepatic artery
Shaking waterbath	Boekel/Grant	8R0534008	Important for mechanical digestion of exocrine tissue
Small surgical scissors	VWR	82027-578	Cuttitng tissue that atached to pancreas