Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

Notes before getting started:

1. Human fetal pancreata were obtained from the Birth Defects Research Laboratory, University of Washington (Seattle, Washington, USA) who harvested the tissue. Informed consent for tissue donation, storage, and use of the samples was obtained from the donors by the center. The protocol consent statement was provided in writing and The University of California, San Diego Human Research Protections Program approved the whole study (Protocol #081237XT).
2. It is essential to maintain both the human fetal pancreas and the container holding the pancreas on ice during the entire procedure. The dissociation and digestion should be performed as fast as possible.
3. Send the clinic where the human fetal pancreas was obtained from the buffer for storage and transport of the pancreas. (RPMI-1640 + 10% normal human serum (NHS), 300 mM trehalose SG, penicillin/streptomycin, and fungizone).

1. Dissection of Human Fetal Pancreas

1. Dissolve 5 mg of RT collagenase XI in HBSS to give a final concentration of 2.5 mg/ml. Filter sterilize the solution with a 0.22 micron syringe filter into a sterile (autoclaved) scintillation vial and store at 4 °C. In a tissue culture hood, set out 2 sterile Petri dishes, a sterile small crucible (if a crucible is unavailable, a small beaker may be substituted), sterilized scissors, and forceps. Add 2 ml HBSS to one Petri dish to wash the pancreas. Aspirate liquid from the tube containing the fetal pancreas, leaving approximately 1 ml.
2. Remove the pancreas with forceps into the 60 mm Petri dish without HBSS. For these experiments, fetal ICCs are generated from fresh pancreata with gestational ages ranging from 9 to 23 weeks. Isolation from pancreata at earlier gestionational ages is difficult due to the size of the pancreas. The protocol is likely applicable to gestational ages beyond 23 weeks, however acquisition of pancreata after this time is difficult because of federal (U.S.) restrictions. Occasionally, the fetal pancreas arrives with splenic, fat, or connective tissue attached from the original dissection. The extra tissue is easily visible to the naked eye and should be removed from the pancreas before starting the protocol.
3. Rinse the pancreas in a minimal volume of cold HBSS (~0.5 ml) in the Petri dish.
4. Move the cleaned pancreas to the small sterile crucible using sterile forceps and place in an ice bucket. Place the crucible in a holder for a 50 ml centrifuge tube and, using 2 pairs of scissors, vigorously slice the pancreas for several minutes. (Usually 2-5 min, but the time depends on the size and firmness of the tissue). Cutting technique is important. Hold one side of the scissors in a fixed position moving only the opposite handles. The tips of the scissors should rest on the bottom of the crucible. Continue with rapid scissor motion to break the pancreas into small pieces. The smaller the pieces of tissue, the better the collagenase will work.
5. Add 1.5 ml of HBSS + the chopped up pancreas to the vial containing the collagenase and place in a water bath shaker set to 37 °C at 200 rpm for up to 10 min. Do not check more frequently than once during the first 5 min. Digestion time depends on the tissue quality, as little as 6 min may be sufficient. The end point is when the particles are small and uniform.
6. Fill the vial (10-15 ml) with cold HBSS to stop the collagenase activity. Place on ice and allow the clumps to settle for 10 min. Often at this point, some cell death has occurred and DNA is released into the media. This can make the media ‘stringy’; in this case, add 100 µl DNase (10 mg/ml stock) to the solution.
7. After the tissue in the scintillation vial has settled for 10 min, aspirate off the top layer leaving it slightly less than half full (7-8 ml). Transfer the cells to a 15 ml conical tube, add 5 ml HBSS. Centrifuge for 5 min at 300 x g.
8. Aspirate the HBSS and resuspend the cells in 5 ml media containing RPMI-1640, glutamax, 10% normal human serum (NHS), 10 mM HEPES pH 7.2, penicillin/streptomycin, and fungizone. Plate on 60 mm Petri dish. For researchers trying to generate β cells, add HGF (10 ng/ml final) to the media. Additionally, a number of groups have demonstrated that supplementing the culture media with the incretin hormone GLP-1 also increases β cells²⁸. Cells should be left in suspension for 72 hr to aggregate and form ICCs.
9. After 72 hr in suspension, cells can be plated on the matrix of the human cell line HTB9 matrix as described previously²⁹. Regardless of growth conditions, the media should be changed every 48 hr.

2. Immunofluorescence of Markers of Endocrine Cell Proliferation and Maturation in ICCs Grown in Suspension, Monolayer, or on Glass Coverslips

1. To measure cell proliferation, BrdU labeling of either adherent cells or cells in suspension is performed for 12 hr prior to PFA fixation. BrdU reagent is diluted 1:100 and filter sterilized in RPMI-1640, glutamax, 10% NHS, 10 mM HEPES pH 7.0, penicillin/streptomycin, and fungizone.
2. For cells in suspension: Centrifuge for 5 min at 300 x g and aspirate culture medium. Add 10 ml PBS to wash the ICCs, centrifuge for 5 min at 300 x g and aspirate. If the ICCs are going to be embedded in paraffin, please follow optional protocol 3.1-3.17. For adherent cells: Remove culture medium and wash cells with PBS as described above. Fix cells for 20 min with 4% PFA at RT, then wash twice with PBS. At this point, the plates can be wrapped with Parafilm and stored in PBS at 4 °C for up to 1 week.
3. Incubate the cells with 0.2% Triton-X 100 in PBS for 10 min at RT. Wash the cells twice with PBS and apply Blocking Buffer (2% donkey serum, 2% bovine serum albumin, 50 mM glycine diluted in PBS) for 1 hr. Wash the cells with Working Buffer (Blocking Buffer diluted 1:10). Dilute the antibody in working buffer. For cells grown on glass coverslips, dilute the antibody in 60 μl total volume. For cells in a 6-well dish, dilute the antibody in 600 µl total volume. To stain multiple epitopes, a cocktail of antibodies may be applied. As a control, use IgG or pre immune serum in place of the specific antibody. Control samples must be incubated with control IgG from the same species as the primary antibody being used.
4. Incubate the primary antibody either 1.5 hr at RT or O/N at 4 °C. Aspirate the primary antibody and wash 3x with Working Buffer at RT.
5. Dilute the secondary antibody at 1:200 for Rhodamine and FITC conjugated antibodies, 1:500 – 1:1,000 for the Alexa conjugated antibodies. It is important to note that all secondary antibodies are light sensitive. Cover the samples in aluminum foil to prevent loss of signal. Incubate for 1 hr at RT.
   OPTIONAL: To visualize the nuclei, DAPI can be added at 1:500 during the secondary incubation. This is useful for quantitation of protein expression in the total cell population.
6. Aspirate the secondary antibody and wash 3x with Working Buffer at RT. For cells grown on a coverslip, gently lift the coverslip with forceps while immersed in the buffer; wash it by immersing in PBS. For cells grown in a 6-well plate, add PBS to wash and aspirate. At this point they are ready to examine under an inverted microscope.
7. Touch the edge of the coverslip gently on a Kimwipe to get rid of excess PBS. Add a drop of mounting gel on a glass slide and invert the coverslip onto the slide. Remove excess mounting gel by aspiration. Tap the coverslip gently with the back of a 200 μl pipet tip to remove bubbles.
8. Dry at room temperature for about 20 min or O/N at 4 °C and examine under a fluorescent or confocal microscope.

3. (Optional) Immunofluorescent Staining of Proteins from Paraffin Embedded ICCs

1. Prepare a 3% agarose solution and let cool to 55-60 °C.
2. Transfer the ICCs incubated in suspension to a 15 ml conical tube, and centrifuge at 1,500 x g for 5 min at room temperature.
3. Aspirate the media from the cells and fix with 500 μl 4% PFA for 10 min at RT. Do not mix the pellet, unless the pellet is very large.
4. Wash the pellet twice with 750 μl PBS. Like the 4% PFA, this waste is considered hazardous and should be disposed of according to your institution's hazardous waste specifications.
5. Gently flick the cell pellet to loosen and add 150-200 μl of the agarose down the wall of the 15 ml conical tube.
6. Mix by gently flicking the tube, but do not form bubbles.
7. Allow to solidify at 4 °C for 5-10 min.
8. Use a 21 G needle to dislodge the pellet and place in a fresh 15 ml conical tube.
9. Paraffin embedding and the preparation of sections on slides can be done on site using a microtome or by your core microscopy facilities.
10. Deparaffinize tissue, hydrate, and wash with water quickly.
11. Add 0.2 M glycine for 30 min at RT to quench the remaining aldehyde.
12. Wash the slide twice with PBS for 2 min each.
13. For antigen retrieval, bring 10 mM Citrate Buffer (pH 6.0) to boil over hot plate. Immerse the slides in the buffer, wait for it to come back to a boil, and wait for 15 min.
14. Remove the beaker from the hotplate. Wait approximately 40 min for the slides to cool in the buffer.
15. Wash the slides twice with H₂O for 5 min each, wash the slides twice with PBS for 5 min each, block with 2% normal donkey serum in PBS for 1 hr.
16. Incubate with primary antibody, diluted to correct concentration in Working Buffer containing 0.2% Triton X-100. Generally, the primary antibody is incubated overnight if staining for transcription factors and for 1.5 hr if staining for hormones, markers of proliferation, and/or differentiation.
17. Follow steps 2.3-2.9 described above.