Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

A. Materials Needed: See Table 1 (reagents) prior to preparation

1. HBSS(P/S): 100 ml 10X HBSS + 900 ml H₂0 + 1% Penicillin/Streptomycin (stored in the refrigerator or kept on ice).
2. 20% BSA stock is made in water and aliquoted and stored at -20 °C.
3. HBSS(BSA): 200 ml 1X HBSS(P/S) + 3 ml of 20% BSA (final BSA concentrations is 0.3%)
4. Islet medium: RPMI + 10% FBS + 1% glutamine + 1% Penicillin/Streptomycin
5. Histopaque 1100: 240 ml 1119 Histopaque + 200 ml 1077 Histopaque
6. 4-0 Suture (McKesson): cut into 2 ¾ inch pieces and stored in a 10 cm Petri dish
7. Instruments: Bend the forceps from Fine Science Tools to 90 °, and file down the serrated teeth of all three pairs of the forceps. The goal is to dull the teeth, not to remove them.
8. Cannula for administration of collagenase/protease mixture: 27G ½ in. needle, 30 ½ in. needle, PE50 tubing cut into 12 in. pieces. File both needles down to a smooth rounded end that has no sharp edges. Insert the 27G needle into one end of a 12 in. piece of PE50 tubing. Remove the 30G needle from the casing by smashing the casing with a pair of pliers, turning the casing ¼ turn each time. Remove the needle with the pliers from the casing and insert it into the other end of the PE50 tubing under a dissection microscope. Be sure to not kink the tubing as you insert the needle into the PE50 tubing. The 27G needle is the end that you will attach the syringe of collagenase.
9. Collagenase (CIzyme MA) and Neutral Protease (CIzyme BP). Reconstitute the TDE per manufacturer’s instructions. Refer to the lot specific Certificate of Analysis to calculate the enzyme activity concentration. Transfer a total of 350,000 collagen degrading activity (CDA) units of the reconstituted collagenase and 100,000 neutral protease units of the reconstituted BP Protease into a conical tube and dilute up to 15 ml total in HBSS (P/S)

B. Step-by-Step Protocol

1. Inflation of Pancreas with Collagenase/Protease Mixture

1. Euthanize mouse by cervical dislocation, saturate the mouse torso with 70% EtOH, open abdominal cavity completely from anus to diaphragm with any dissection scissors and forceps.
2. Place the mouse on the platform of a dissection microscope.
3. Pull the caecum and ascending colon out and set them outside the body cavity to your left. See Figure 1 for a summary of the following steps.
4. Position the lobes of the liver against the diaphragm; they should remain there if the body cavity is open far enough. Find the hepatic artery, portal vein and bile duct bundle leading into the liver by gripping the duodenum with curved forceps. With another pair of curved forceps, reach under the artery, vein, and duct bundle and pull a 2¾ in. piece of 4-0 suture under the artery, vein, bile duct and tie it once and pull it tight as close to the liver as possible.
5. Using two pairs of curved forceps, carefully grab the duodenum and find the bile duct attached to the duodenum at the papilla. Use a tong of the forceps to poke through the pancreas and connective tissue right under the bile duct close to the papilla. Poke your forceps quickly through the organ to make a hole and then put both tongs of forceps through and grab another 2¾ inch piece of 4-0 suture through and tie it once and pull it to a small circle, but do NOT pull tight.
6. Change to the 90 ° forceps and to the Superfine Vannas scissors. With the 90 ° forceps, carefully pinch and pull the small intestine until the bile duct is taut. Make one horizontal cut across the papilla with the scissors by stabbing the open scissors into the intestine and then cutting with one motion. Try to only make one cut across the papilla without dislodging the bile duct from the papilla.
7. While still holding the intestine with the 90 ° forceps, insert the cannula into the bile duct up to half the length of the bile duct, and then gently pull the suture tight around the cannula.
8. Fill a 3 ml syringe with 2.0 ml of collagenase/protease mixture and inject into the cannula at a slow, constant pace.
9. After inflation of the pancreas is complete, remove cannula from bile duct and use the curved forceps and curved spring scissors to remove the inflated pancreas by starting at the descending colon and snipping the connective tissue as you pull the intestines up. Continue to remove the pancreas from the intestines until you reach the bile duct, then remove the pancreas from the spleen, then the stomach and then from the viscera of the abdominal cavity. Finish removing by snipping away the sutures. Add the pancreas to a 50 ml tube containing 5 ml of HBSS(P/S). This tube should be on ice.
10. Repeat the procedure above for each animal up to four animals. For optimal results, dissociate only four pancreata at one time. Typically, while the first four are in the 37 °C water bath, we inflate another four pancreata.

2. Pancreas Dissociation

1. Place the 50 ml tubes containing the pancreata into a 37 °C water bath for 15 min.
2. Gently rock the tube and check for tissue dissociation, be sure tissue comes apart easily, then swirl the tubes 12 times while you are holding the tube by the lid.
3. Add no more than 30 ml of HBSS(BSA) (this can be a lower amount, depending on how much is required to balance the tubes for the centrifugation step), and centrifuge at 290 x g for one min.
4. Aspirate supernatant carefully and leave a small amount of supernatant (2-3 ml) at the bottom so as to not disrupt the cell pellet.
5. Using a 30 ml syringe attached to 14G needle, add no more than 10 ml of HBSS(BSA) and aspirate the suspension up and down 2 times.
6. Filter the cell mixture through a plastic tea strainer into a 50 ml tube and rinse with another 10 ml HBSS(BSA).
7. Centrifuge at 330 x g for 2 min.
8. Decant supernatant and invert tubes to drain on an absorbent paper towel for 1 min.
9. Resuspend each tube in 10 ml cold 1100 histopaque.
10. Overlay the histopaque with 10 ml HBSS(BSA) and centrifuge at 900 x g for 18 min.
11. Remove the entire 20 ml of supernatant using a large bore 25 ml pipet and pass the supernatant through an inverted 70 μm filter.
12. Rinse the islets into a 10 cm Petri dish by pipetting 10 ml islet media through the filter while holding it right side up over the dish.
13. Culture the islets in a 37 °C, 5% CO₂ tissue culture incubator until ready for experimentation. Typically, islets are hand-picked and counted prior to experimentation.

3. Representative Results

Islet yields, morphology, and quality are the general parameters used to judge the success of islet isolation. In our hands, islet yields from the average C57BL/6 mouse are in the range of 150-250 islets using this protocol (see Table 2), and are comparable to data obtained using optimized Sigma type XI collagenase. However, yields can vary depending upon the mouse strain used and the age of the mouse. Most of the animals we employ are in the 8-16 week age range This protocol has been tested on multiple mouse strains, including C57BL/6 (180-250 islets/mouse), CD1 (200-300 islets/mouse), 129/B6 (200-300 islets/mouse), BLKS-db/db (120-200 islets/mouse), NOD (10 week-old, 100-150 islets/mouse), and NOD-SCID (100-150 islets/mouse). Typical islet morphology is shown in Figure 2, in which islets have a round to oblong shape with relatively uniform size (although size uniformity can vary from strain to strain). Figure 3 shows our typical analysis of glucose-stimulated insulin secretion from C57BL/6 mouse islets isolated using purified TDEs vs. Sigma type XI collagenase. Typically, a high quality islet preparation results in an insulin stimulation at 25 mM glucose that is 6-20 times greater than that observed at 2.5 mM glucose. Other applications can also be used to test the quality of isolated islets, and these include use of perifusion techniques, Ca²⁺ imaging (with Fura2), and reverse-transcription PCR, among others¹¹.

Figure 1. A summary of the cannulation of the bile duct.

Figure 2. Typical appearance of C57BL/6 islets at 24 hr following isolation. Images were acquired on an inverted microscope at 40X magnification.

Figure 3. Glucose-stimulated insulin secretion of C57BL/6 islets. At 24 hr following isolation, 100 islets were incubated in a 12-well dish for 1 hr at 37 °C in Krebs-Ringer HEPES-buffered solution containing 2.5 mM glucose. Islets were then transferred to Krebs-Ringer HEPES at 2.5 mM glucose for an additional hr, after which the supernatant was collected for insulin measurement using a two-site immunospecific enzyme-linked immunosorbent assay (ELISA) (Crystal Chem). The same islets were subsequently transferred to Krebs-Ringer HEPES-buffered solution containing 25 mM glucose, and insulin release into the medium was measured by ELISA after 1 hr of incubation.