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.
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.
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 solution1,2,3. 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 itself1,4,5. 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 islets3,7. 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 others1,4. 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 mechanically1,4,7. 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 islets1,10. 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 tissue8,11. 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.
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
2. Preparation
3. Procedure
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 1 mouse.
In this paper, a number of very realistic images are included: Figure 3 shows the abdominal cavity of the mouse, exposing the common bile duct and hepatic artery. Figure 4 shows the entire length of common bile duct, which appears to be a lighter color, as well as the ampulla of Vater, which is bigger and shinier near the juncture (where the needle will be inserted) between the pancreas and duodenum. A clamp is placed at the common bile duct and hepatic artery bundle close to the liver to block off the flow of collagenase P into liver. Failure to clamp the bile duct correctly or tight enough will result in leakage and incomplete perfusion of the pancreas. Figure 5 shows the needle inserted at the ampulla of Vater into the common bile duct. Once the needle is in the common duct, forceps are used to stabilize the needle to prevent it from puncturing the duct while injecting. Once the injection begins, the pancreas will begin to swell from proximal end to distal end; the splenic region should begin to inflate after about 1 mL of collagenase injection. Backflow into the intestines can lead to an undesirable inflation of the duodenum; this can be remedied by readjusting the placement of the needle (pull it out a little, or reinsert slightly deeper) and by properly stabilizing the needle using forceps. The injection is considered a success if all regions of the pancreas are inflated (duodenal, gastric, and splenic lobes) as shown in Figure 6. Removal of the pancreas should begin from the splenic region. The inflated pancreas is chopped into chunks using fine surgical scissors (Figure 7A). After the 12–13 min digestion and mixing by hand-shaking, the tissue-containing suspension appears more homogeneous (Figure 7B). Subsequently, the islets are purified by the density gradient after centrifugation. Figure 7C shows that an islet suspension layer is formed between HBSS and the density gradient after centrifugation. Good/healthy islets appear as smooth round-shaped; bad/damaged islets show rough edges, and undigested exocrine tissue shows irregular shape and appears more translucent as shown in Figure 8.
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.
Figure 2: Schematic illustration of the protocol.
The most critical steps of this procedure are the clamping of the common bile duct near the liver, and injecting collagenase P via the ampulla of Vater into common bile duct to digest the pancreas. Please click here to view a larger version of this figure.
Figure 3: Location of common bile duct.
Forceps hold up the common bile duct and hepatic artery bundle. Please click here to view a larger version of this figure.
Figure 4: Illustration of common bile duct and ampulla of Vater.
A clamp is placed on the common bile duct and hepatic artery bundle near the liver. The black arrows point to the ampulla of Vater where the needle will be inserted. Please click here to view a larger version of this figure.
Figure 5: Cannulation of common bile duct.
After proper cannulation of the common bile duct, the ampulla of Vater is injected with trypan blue (for demonstration purpose only) to better emphasize the placement of the common bile duct. Please click here to view a larger version of this figure.
Figure 6: Fully inflated pancreas.
The dotted line shows the boundary of the fully perfused pancreas. Forceps hold up the splenic region where islets are most concentrated. Please click here to view a larger version of this figure.
Figure 7: Islet isolation and purification steps.
(A) Mechanically chopped tissue pieces of pancreas before digestion. (B) Digested pancreas—homogeneous tissue suspension of collagenase-perfused pancreas after 13 min of incubation in a shaking water bath at 37 °C, followed by 30 s of mixing by hand. (C) Islet suspension layer is formed between the HBSS and the density gradient after centrifugation. Please click here to view a larger version of this figure.
Figure 8: Representative islet images from Fluorescence Cell-Imager.
(A) Good islets, bad islets and exocrine tissue are seen. (B) A cluster of good islets is shown. (C) Panel shows undigested exocrine tissue attached to a good islet. Good/healthy islets show smooth round edges, indicated by the red letter “G”; bad/damaged islets show irregular shape and rough edges, and are indicated by blue letter “B”. Undigested exocrine tissues appear translucent, often attached to islets, and are indicated by green letter “E”. Please click here to view a larger version of this figure.
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 pancreas1,4,7. 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 complete digestion of the splenic region, which has the highest concentration of islets4,6. This protocol, with well-controlled digestion time and carefully executed purification steps, can produce 250–350 healthy islets. The islets isolated from this protocol have been used successfully to study glucose-stimulated insulin secretion ex vivo12. From our experience, insulin secretion increased 4-fold upon high glucose stimulation (22.2 mM) compared to baseline (3.3 mM) after overnight incubation in 5.5 mM glucose-containing RPMI-1640 complete media. The survivability/functionality of the islets under prolonged incubation (2–7 days) has not been tested.
Even though the presented protocol includes detailed notes and visual demonstrations, some adjustments are needed to achieve the optimal conditions for high yield and high-quality islets. Two most common problems that may impede the success are improper cannulation of the ampulla of Vater or accidental puncture of the duct. To avoid these, one should ensure that the site of penetration is precisely where the ampulla meets with the duodenum. This area is bigger and relatively easy to identify, which allows for multiple punctures without seriously compromising the integrity of the duct. Once within the ampulla, the orientation of the needle should be parallel with the duct rather than angled. At this point, push the needle into the duct for about 1/4 of the duct’s length, and then stabilize the needle with forceps while slowly injecting collagenase; this will help to prevent the needle from bending under the pressure and accidentally puncturing the duct. Other problems may include over- or under-digestion of the pancreas; this may require modification based on age, strain and sex of the mice. Damaged islets due to over-digestion (enzymatic and mechanical) or prolonged exposure to the density gradient may occur. These are common problems that exist for similar methods; some minor adjustments should yield significant improvements. A suggestion when dealing with these types of issues is to choose one variable to modify at a time (e.g., time of digestion or concentration of collagenase).
The main advantage of this protocol is the route of enzyme delivery: having collagenase P to directly digest the exocrine pancreas using an easier anatomical route which increases digestion efficiency1. This method has been reported to yield a 50% increase in number of islets compared to the method of excising the pancreas, chopping it, and exposing it to collagenase13. This protocol only requires the use of a single density gradient, making it significantly less labor-intensive and more cost-effective, as compared to other methods which require the preparation of multiple gradients at different densities, or the complex Percoll method that requires additional time1,8,11. The gradient method employed in this protocol has also be used in islet isolation by others4. This method of islet isolation provides the scientist with an improved tool for studying pancreatic islets. Future applications of this protocol include alteration to enhance the efficacy when dealing with diabetic mice. As Do et al. have observed, diabetic mice, depending on glucose levels, yield fewer islets (less than 100), with reduced size and appearance of islets4. We have observed this phenomenon and believe that diabetic islets are more vulnerable to enzymatic and mechanical digestion which require special care. Additionally, islet density may alter its appearance in the density gradient, further optimization of the protocol would help to increase the yield of diabetic islets.
The authors have nothing to disclose.
We are extremely grateful to Ms. Jennifer Munguia for her artistic illustration of the schematic diagram. We thank Mr. Michael R. Honig at Houston’s Community Public Radio Station KPFT for his editorial assistance. This study was supported by American Diabetes Association #1-15-BS-177 (YS), and NIH R56DK118334/R01DK118334 (YS). This work was also supported by the USDA National Institute of Food and Agriculture, Hatch project 1010840 (YS) and R01 DK095118 (SG).
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 |