Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells
Published: April 30, 2023
Abstract
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 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).<…
Representative Results
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. Please click here to view a larger version of this figure. <p class="jove_c…
Disclosures
The authors have nothing to disclose.
Materials
| 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 moisture barrier |
VWR | 82020-845 | To absorb blood from surgical procedures |
| 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 declaration 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 |
| 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 | Cutting tissue that attached to pancreas |
| RNaseZap | Fisher-Scientific | AM9780 | For removing RNase |
| Isoflurane | Piramal | B13B16A | To anaesthetize mice prior surgery |
Cite This Article
Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells. J. Vis. Exp. (Pending Publication), e20311, doi: (2023).