Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

Samenvatting

Source: Villarreal D. et al. A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice. J. Vis. Exp. (2019)

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. This protocol explains a method for isolating pancreatic islets by first perfusing collagenase via Ampulla of Vater to digest the exocrine pancreatic tissue and a simplified density gradient centrifugation to purify the islets from mice.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

The surgical tools needed are 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 ....

Resultaten

Figure 1: Surgical tools.
Curved surgical scissors, cover glass forceps, micro Adson forceps, curved forceps, small surgical scissors, and Schwartz micro serrefines (microvascular clamp) are shown.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
3 mL syringe 	BD 	309657	Holding 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
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<br/> moisture barrier	VWR	&nbsp;82020-845&nbsp;	To absorb blood from surgical procedures
Centrifuge 5810R with swing<br/> bucket and deceleration capability	Eppendorf&nbsp;	5811FJ478114&nbsp;	Use for pelleting tissues, pellet is formed at bottom of conical tube - swing bucket centrifuge is needed. Also the declaration feature is important to form the gradient layers
Collagenase P- 1g&nbsp;	Roche Diagnostics&nbsp;	11249002001	For digestion of exocrine pancreas
Curved surgical scissors&nbsp;	Fisher-Scientific&nbsp;	13-804-21&nbsp;	For cutting open mouse abdomen
Dissection microscope&nbsp;	Olympus&nbsp;	SZX16&nbsp;	Used for identification of key anatomical structures to accurately<br/> deliver collagenase into pancreas
Hank's Balanced Salt Solution 10x	Corning&nbsp;	20-023-CV&nbsp;	Washing cells
Histopaque-1077&nbsp;	Sigma&nbsp;	RNBF5100&nbsp;	For gradient formation
Light source&nbsp;	Leeds&nbsp;	LR92240&nbsp;	Enhancing visibility of microscope
RPMI-1640 Media w/o L-Glutamine&nbsp;	Corning&nbsp;	15-040-CV&nbsp;	Culturing Islets
Schwartz micro serrefines (Microvascular clamp)&nbsp;	Fine Science Tools&nbsp;	18052-01	Clamping common bile duct and<br/> hepatic artery
Shaking waterbath	&nbsp;Boekel/Grant&nbsp;	8R0534008	Important for mechanical digestion of exocrine tissue
Small surgical scissors&nbsp;	VWR&nbsp;	82027-578&nbsp;	Cutting tissue that attached to pancreas
RNaseZap&nbsp;	Fisher-Scientific	&nbsp;AM9780&nbsp;	For removing RNase
Isoflurane&nbsp;	Piramal&nbsp;	B13B16A&nbsp;	To anaesthetize mice prior surgery