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^1,4,5. It is known that islets are not evenly distributed in the pancreas; the splenic region contains the most islets⁶. While the second method using anatomical routes to deliver digestive enzymes allows for a more complete perfusion of the pancreas, including the splenic region, this procedure often requires clamping or suturing of the ampulla of Vater that is technically challenging. In terms of islet purification, multiple density gradients, as well as cell strainers and magnetic retraction have been used to purify the islets^3,7. The utilization of these gradients can be time consuming and the Ficoll gradients can result in toxic damage of islets⁸.

The current protocol is built on the method described by Li et al.⁷, with additional modifications added based on the experience of ourselves and others^1,4. The most critical steps of our protocol are clamping of the common bile duct near the liver end, injecting collagenase P via the ampulla of Vater to digest the exocrine tissue, and then using a shaking water bath to expedite the digestion mechanically^1,4,7. Subsequently, a 'STOP' solution is applied to inhibit further digestion of the islets; HBSS is used to wash off the remaining collagenase P and STOP solution. When the Ficoll method was used to purify human islets, yield was reported to be twice the islets with greater functional capability (e.g., insulin secretion) as compared to the use of Percoll gradients⁹. However, studies have questioned the use of Ficoll gradient due to its toxic effect on the islets^1,10. It has been reported that the Histopaque gradient provides optimal purification kinetics for mouse islet isolation, which produces good yield of high quality islets with simpler steps and lower cost¹. In our protocol, Histopaque-1077 is used to purify islets from other residual tissue^8,11. The harvested islets can be cultured in complete RPMI-1640 media, or directly utilized in RNA/protein quantitation.

Our protocol, using a combination of collagenase P digestion and a single gradient purification step, is simpler than other published protocols. Our method does not require demanding surgical procedures and has just a few simple steps. More importantly, this protocol consistently produces a good yield of high quality functional islets (250-350/mouse) as we reported¹².

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 solution. Keep the STOP solution at 4 °C.
3. Prepare collagenase P solution (must be made fresh within 1 h of being used) by adding 1 mg/mL of collagenase P to ice cold 1X HBSS; use 6 mL/mouse.
   1. Add 0.05% (w/v) bovine serum albumin (BSA) to collagenase P solution (this provides nutrients to the isolated islets). For example, use 3 mg BSA in 6 mL collagenase P solution. Keep on ice.
   2. Calculate and prepare the amount of collagenase P needed for all mice in one 50 mL tube.
4. Prepare complete RPMI 1640 medium by adding 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, INS-1 cell supplement (10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol) to 500 mL RPMI 1640 media containing 5.5 mM glucose to be used for overnight culture and incubation.

2. Preparation

1. Fill three 50 mL tubes of 25 mL RNase inhibiting solution, 70% ethanol or distilled H₂O. These solutions will be used for cleaning surgical tools prior and during the procedure.
2. Soak the tips of the tools in RNase inhibiting solution for 30 min before starting the protocol; this eliminates any potential RNase on the tools.
3. Pre-cut absorbent pads to 6 in x 6 in size for use during surgery.
4. Prepare 1X HBSS solution in advance and store at 4 °C. Prepare STOP solution prior to surgery. Store at 4 °C.
5. Prepare collagenase P solution immediately prior to surgery and store on ice.
   NOTE: This should be used within 2 h of preparation.
6. Label 50 mL tubes for digestion and purification; prepare 2 tubes for each mouse, one for digestion and the other for islet purification. Ensure that the animal ID is on both tubes.
7. Add 3 mL of collagenase P solution into the first 50 mL tube. The remaining 3 mL of collagenase P will be injected.
8. Draw the remaining 3 mL of collagenase solution into a 3 mL syringe mounted with 30 G ½ in needle. Place the syringe on ice.

3. Procedure

1. Remove all the tools from RNase inhibiting solution, then dip them first in the tube with 70% ethanol, then in the tube containing distilled H₂O, then air-dry on clean paper towel.
2. Place the mouse in a chamber containing 0.5 mL of isoflurane until mouse is deeply anaesthetized.
3. Remove mouse from the chamber and check the state of anesthesia by pinching a foot pad with forceps. Deep anesthetization is based on the observation that breathing become steadily slow and mouse is unreactive to foot pinching. After confirming that the mouse is deeply anaesthetized, euthanize the mouse with cervical dislocation, and then place the mouse on the absorbent pad.
   1. Place anaesthetized mouse on its stomach, applying pressure to the neck and dislocating the spinal column from the brain by pulling tail.
4. Tape the limbs of the mouse in supine position to the absorbent pad, spray the body with 70% ethanol, and wipe excess off the excess ethanol.
5. Use cover glass forceps and curved surgical scissors to make incisions. First make a horizontal incision on the skin of the abdominal area (~3 cm), pull the skin wide open to expose the abdominal wall. Then make a vertical incision (~3–4cm) on the abdominal peritoneum to fully expose the pancreas in abdominal cavity (Figure 3).
6. Push the lobes of the liver superiorly to expose the bile duct, it will appear as a pale pink tube (Figure 3).
7. Carefully move the intestines from the right lumbar/iliac region of the abdominal cavity to the right, exposing the bile duct and hepatic artery (Figure 3).
8. Carefully clamp the common bile duct using the Schwartz micro serrefines (Figure 1) as close to the liver as possible.
9. Identify the ampulla of Vater, which is located at the duodenal papilla, formed by the union of the pancreatic duct and the common bile duct. The ampulla of Vater appears swollen when viewed under a dissection microscope which is the entry point to the common bile duct (Figure 4).
   NOTE: Adjusting intensity/angle of light of the dissection microscope can make it easier to locate.
10. Insert the syringe with 3 mL of the collagenase P solution into the ampulla of Vater. Push the needle into the duct for about 1/4 of the length of the common bile duct (ampulla leads into duct) as shown in Figure 5.
11. Once the needle is in the ampulla, ensure that the orientation of the needle is such that it is parallel with the duct.
12. Stabilize the needle by clamping with micro Adson forceps to prevent it from puncturing the duct (Figure 5).
13. Slowly and steadily inject 3 mL of the collagenase P solution from the syringe into the common bile duct (enough to feel resistance) as shown in Figure 6. The goal is to create backflow pressure to force the collagenase to enter the pancreatic duct. Injection is considered successful if the head, neck, body and tail region of the pancreas are all fully inflated.
    NOTE: Islet yield will be low if either the pancreas is not fully inflated, or the splenic area is not fully inflated. The splenic area contains the highest number of islets⁶. Inflation can be confirmed by the appearance of open spaces between pancreatic tissue that are filled with solution.
14. Carefully dissect out the inflated pancreas and place it in a 50 mL digestion tube containing 3 mL of ice-cold collagenase P solution.
    1. Remove the pancreas using 2 forceps (curved and Micro Adson; See Figure 1): (starting from the spleen, pull the pancreas away from spleen and continue removing from the stomach and along the duodenum.
       NOTE: No incisions required.
    2. Chop pancreas for 3–5 s in the digestion tube with 3 mL of ice-cold collagenase P solution using fine surgical scissors (Figure 7A).
15. Secure the tube to a rack in 37 °C water bath and shake at 100–120 rpm for approximately 12–13 min.
16. After incubation, gently shake the tubes by hand to disrupt the tissue until the collagenase P digestion solution becomes homogenous (Figure 7B). Homogeneity is confirmed by a sand-like appearance of fine particles of pancreas. About 30 s of gentle hand-shaking is usually enough. Hold the tube up to light to examine if the tissue is well homogenized; shake for another ~15 s if needed.
17. Once digested, immediately place the tubes on ice and add 40 mL of ice-cold STOP solution to terminate the enzymatic digestion.
    NOTE: At this point digestion tubes can be left on ice up to 2 h, if working on multiple mice.
18. Centrifuge the tube in a swinging-bucket centrifuge at 300 x g for 30 s (temperature of centrifuge is flexible).
    NOTE: Use a swinging-bucket centrifuge so that the tissue pellet is formed at the bottom of the 50 mL tube and not on the wall of the tube; this is critical for better islet yield.
19. Decant and repeat the centrifugation with STOP solution 2 more times, decanting the solution after each spin. Use 20 mL of STOP solution for each subsequent wash.
    1. Before each spin, disrupt the pellet by gently shaking the tissue pellet in the 50 mL tube in 20 mL STOP solution.
20. Re-suspend the tissue pellet with 40 mL of ice cold HBSS and centrifuge at 300 x g for 30 s.
21. Decant and repeat the centrifugation using a swinging-bucket centrifuge with HBSS solution two more times, decanting the solution after each spin. Use 20 mL of HBSS for each wash.
    1. Before each spin, disrupt the pellet, to detach it from the bottom of the tube by gently shaking the tube containing 20 mL HBSS solution.
22. After the last centrifugation remove all HBSS.
23. Then add 5 mL of room temperature density gradient to the 50 mL tube containing the pellet. Vortex briefly at low speed until homogenized.
24. Add another 5 mL of the room temperature density gradient to the 50 mL tube. Do not vortex/mix.
    NOTE: It is critical to remain steady and still so as to allow better gradient to form without disruption.
25. Pipette 10 mL of room temperature HBSS buffer into the tube (containing the density gradient), drop-by-drop gently and slowly to allow a gradient to form. Use a pipette gun with a 10 mL pipette to add HBSS dropwise.
26. Using a swinging-bucket centrifuge, spin tubes at 1700 x g for 15 min. Make sure to change the speed of both acceleration/deceleration to the lowest setting to maintain the gradient (Figure 7C).
27. After the spin, carefully remove the tubes without disturbing the gradient. Using a pipette gun pre-wetted with cold HBSS (pipette HBSS up and down), pipette out the layer of islets (5–10 mL) formed between the density gradient and HBSS into the new 50 mL islet collection tube.
    NOTE: It is helpful to wet the pipette with cold HBSS prior to pipetting the islets to prevent the islets from sticking to the inner walls of the pipette.
    1. In case the separation is incomplete, islets would be visible in the density gradient layer, pipette out the entire 10 mL of the density gradient (bottom layer) along with the islet layer formed at the interface (only to leave about 8–10 mL of the HBSS top layer behind).
       NOTE: Collecting both the islet layer and the density gradient layer (bottom layer) can result in the collection of debris which may lengthen the islet picking time, but no other steps will be altered. Collecting both these layers is not necessary when islet layer is well formed.
28. Add 20 mL of ice cold HBSS to the new 50 mL tube containing islets, then centrifuge at 350 x g for 3 min in a swinging-bucket centrifuge.
29. After the spin, carefully pipette (do not decant) out the supernatant (leave ~3 mL at the bottom) without disturbing the pellet containing the islets at the bottom. Discard the supernatant.
30. Repeat washing and centrifuging at least 3 times. Add 20 mL of HBSS each time, and be sure to suspend the pellet before each spin.
31. Warm the RPMI-1640 complete media bottle in a water bath at 37 °C prior to use. After the last centrifugation, remove all of the HBSS and add 4 mL of previously prepared complete RPMI-1640 media (containing FBS, INS-1 cell supplement and penicillin/streptomycin) to the pellet.
32. Dislodge the pellet by gently swirling the tube and immediately pour the RPMI-1640 media into a 100 mm Petri dish. Add another 5 mL of RPMI-1640 media to the tube and gently swirl it to wash off any remaining islets, then also pour the media into the same Petri dish.
    1. Under a dissection microscope, pick healthy islets from the Petri dish using a 20 µL pipette, and put them in a new Petri dish containing 10 mL of complete RPMI 1640 media. When viewed under a dissection microscope, islets should appear spherical/oblong and golden-brown color with a smooth surface compared to the relatively transparent, wispy exocrine tissue.
       NOTE: Magnification of dissection microscope is usually set at 12.5–16x. Islet yield will vary depending on various factors including strain, age and sex of mouse. This protocol usually yields 250–350 healthy islets from healthy C57BL/6J mouse age 4–10 months old.
33. Incubate the islets in a sterile incubator at 37 °C with 5% CO₂ infusion and 95% humidified air overnight for experiments the next day, or freeze the islets for desired analysis at a later time.
    1. Once islets are frozen, they cannot be used for secretion assays, only RNA or protein quantification. To collect cells to freeze, place them in HBSS buffer, collect all islets in 200–500 µL of buffer, transfer to 1.5 mL tube, centrifuge 350 x g for 1–2 min, remove the supernatant leaving no more than 30–40 µL solution, place in -20 °C for short term storage (several days) or -80 °C for long term storage.

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