Islet Purification from Rodent Pancreas

The same general protocol is used for the purification of islets from mouse and rat pancreata. All media used for islet isolation should be equilibrated to room temperature except the HBSS. Note that this entire procedure, excluding centrifugation and digestion, should be performed using sterile technique.

1. A 5-rat or 25-mouse preparation will require a minimum of 500 mL HBSS, a maximum of 500 mL cCMRL-1066, Ficoll dilutions (20 mL of 25% dilution and 10 mL of 23%, 20.5%, and 11% dilutions), and collagenase (one preweighed volume per tube).

2. Begin by placing the isolated pancreata into the evaporating dish. Using both pairs of sterile straight iris scissors, chop the tissue into small evenly sized pieces (see Fig. 2) to ensure even and consistent digestion.

3. Wash the minced pancreatic tissue using HBSS two to three times. This can be accomplished by quickly pouring off the HBSS and refilling the evaporating dish with fresh HBSS. Allow the tissue to settle to the bottom for 5-10 s between washes. Pancreatic tissue should sink, and the adipose tissue that floats should be discarded.

4. Using a siliconized sterile Pasteur pipet that has been cut to remove the narrow tip, transfer the pancreatic tissue from the evaporating dish and evenly distribute the tissue into sterile, siliconized 16 X 100 glass culture tubes (tissue must be distributed evenly in the test tubes for proper digestion). The average tissue volume per tube

Chopped pancreata i

Pancreatic Islets Purification
Fig. 2. Preparation of pancreata for collagenase digestion. The chopped pancreata in this evaporating dish demonstrate the small, even size of tissue fragments ideal for optimum collagenase digestion.

should be approx 3 mL. For ease of preparation, one test tube holds the equivalent of one rat or five mouse pancreata.

5. Allow the tissue to settle to the bottom of the tubes for 5-10 s. Using the same Pasteur pipet, remove as much HBSS as possible from the top of the tissue. There should be approx 1 mm of media remaining on the top of the tissue.

6. Quickly add the premeasured collagenase to each tube, plug tubes with sterile rubber stoppers, and use Parafilm® strips to secure the stoppers in the tubes (see Notes 1 and 2).

7. Place the tubes into the wrist-action shaker clamps (which are set to shake the tubes horizontally) submerged into a 38-39°C water bath. Be sure that the shaker is set at the maximum arc and turn the timer to the hold position. Allow the tubes to shake for the appropriate amount of time as determined for each lot of collagenase (see Note 3).

8. Once digestion is complete, stop the collagenase reaction by quickly pouring approx 8 mL cold HBSS into the test tubes (see Note 4).

Shake the tubes vigorously by hand to dilute the collagenase solution and pellet the tissue by centrifugation. This is accomplished by bringing the centrifuge up to 805g and then immediately stopping the spin with the brake engaged. Quickly decant the supernatant and repeat two additional times as outlined in step 9, bringing the centrifuge up to 453g each time. After the last spin, before decanting the supernatant, remove the foam layer on the top of the media with a standard Pasteur pipet. Then, quickly decant the supernatant and remove the last drop of media from the tube with the Pasteur pipet.

Add 4 mL of 25% Ficoll to each tube using a disposable pipet, and vortex the tube at approximately three-quarters speed. Using the Pasteur pipet, gently remove any mucin from the mixture. Mucin is the byproduct of the collagenase digestion, which appears as a gelatinous body that should be removed from the tissue mixture. To remove it, gently swirl a Pasteur pipet in the mixture. The mucin will adhere to the pipet and can be discarded (see Fig. 3). Note that mucin will not always be present in each digestion and can vary from tube to tube. Once the mucin is removed, prepare a Ficoll step gradient by slowly layering 2 mL 23% Ficoll, 2 mL of the 20.5% Ficoll, and 2 mL of 11% Ficoll to each tube (see Fig. 4) (see Note 5). Spin the tubes at 800g for 12 min at room temperature with no brake.

Once the spin has completely stopped, return the tubes to the hood. Using the Pasteur pipet, remove islets from the 11-20.5% interface and place into one to two sterile 15-mL-thick-walled glass conical tubes containing 2 mL HBSS. Repeat this procedure for the 20.5-23% interface and place islets into one or two separate conical tubes. Following transfer of material at each interface, fill each conical tubes with HBSS to a final volume of approx 12 mL. Resuspend the pellet by pipetting up and down with a Pasteur pipet until the Ficoll is completely mixed with the HBSS. Centrifuge the tubes at 805g for 20-30 s and stop with the brake. Decant the supernatant and repeat this procedure two additional times.

Add 6 mL cCMRL-1066 to the pellet and resuspend the islets using a Pasteur pipet. Spin the tubes for 5 s (including acceleration time) and immediately stop the spin. Decant the supernatant of each tube into a separate 60 X 15-mm Petri dish and save. Repeat this washing step two more times, decreasing the centrifugation time by 1 s for each wash.

Once the washes are complete, add 4 mL CMRL media to each tube, and using the pipet, transfer the remaining pellet into a separate Petri dish. Using a flame-pulled Pasteur pipet and dissecting microscope, remove all of the duct and acinar tissue that remain in each dish. This can be accomplished by either selectively moving the islets to new, clean Petri dishes or swirling the plate and sucking off the acinar and ducts and discarding them into a waste container. Replace cCMRL-1066 as needed during the cleaning process. The preparation should be free of as much extraneous tissues as possible to ensure optimum islet culture conditions. Once the preparation is free of all acinar and ductal tissues, divide the total pooled islets (300-600 islets/rat or 80-180 islets/mouse) into four fresh 60 X 15-mm Petri digested tissue in 25% ficoll mucin

Ficoll Gradient Islet
Fig. 3. Removal of mucin. Mucin is a byproduct of the collagenase digestion. It is important to remove this byproduct from the remaining pancreatic tissue prior to Ficoll gradient centrifugation.

dishes. There should be no more than 300 islets per dish for optimum culture conditions. Remove all media and add 2-2.5 mL fresh cCMRL-1066 per Petri dish. The islets can now be cultured at 37°C with 5% CO2 for 1-3 d. If a longer culture time is desired, the media should be replaced after 3 d (see Notes 6 and 7).

0 0


  • uffo lightfoot
    How to stop collagenase digestion?
    6 years ago

Post a comment