Animal models

Mouse Pancreatic Epithelial Cell Isolation and Spheroid Culture

1. Prepare G Solution: HBSS (Gibco) + Glucose (5 mg/ml) (Sigma-Aldrich) + Pen/Strep (1X) (Gibco).  
   • G Solution: Dissolve 2.5g Glucose in 10 ml HBSS (from a 500ml bottle). Pass the solution through a syringe filter back into the 500ml bottle to sterilize. Add 5ml of 100x Pen/Strep into the 500ml bottle.
2. Harvest one pancreas into ~10 ml G solution in a falcon tube on ice.
3. In the hood: aspirate G solution and transfer the pancreas into a 10cm dish.
4. Cut the pancreas with blades into small pieces (resulting in pieces ~1mm in size).
5. Resuspend the cut tissue in ~10 ml G solution in a in a fresh falcon tube.
6. Allow tissue to settle (for ~3 min) and aspirate G solution and any floating fatty tissue. Repeat G solution wash again.
7. Prepare Collagenase IV/Dispase II solution: PDEC medium + Collagenase IV (2 mg/ml)(Gibco) + Dispase II (2 mg/ml)(Roche) followed by filter sterilization.  
   • PDEC medium: DMEM/F12 1:1 supplemented with 2.5 mM L-Glutamine, 15 mM HEPES Buffer (HyClone), 5 mg/ml Dglucose (Sigma Aldrich), 1.22 mg/ml nicotinamide (Sigma Aldrich), 5 nM 3,3,5-Tri-iodo-L-thyronine (Sigma Aldrich), 0.5 μM hydrocortisone Solution (Sigma Aldrich), 100 ng/ml cholera toxin (Sigma Aldrich), 0.5% insulin-transferrin-selenium (Corning), 100 μg/ml penicillin/streptomycin (Gibco), 0.1 mg/ml soybean trypsin inhibitor (Sigma Aldrich), 20 ng/ml EGF (Peprotech), 25 μg/ml bovine pituitary extract (Invitrogen), and 5% Nu Serum IV Culture Supplement (BD).
8. Resuspend the tissue in Collagenase IV/Dispase II solution (10 ml per pancreas).
9. Digest tissue with constant stirring at 37°C for 45 min in an incubator. Meanwhile, prepare collagen-coated plates or gelatin-coated plates. Pipetting the tissue several times every 10 minutes to help break up all big tissues. Tissue should be free from large chunks at the end of digestion.
   • Collagen-coated TC plates
   Prepare plates with rat tail collagen type I (at 2.31 mg/ml final concentration) (BD). Calculate volumes needed (see individual bottle labels – each batch is different in concentration).
   For thick collagen: 1.5ml/well for a 6-well plate, 3ml/6cm dish, and 5-6ml/10cm-dish.
   For thin collagen: 0.5 ml/well for a 6-well plate, 1ml/6cm dish, and 2ml/10cm-dish.
   Volume of Collagen (Vc) = Total volume (Vt) X 2.31 / concentration labeled on the bottle
   In a 50ml falcon tube, add following on ice:
   - 10 X PBS containing PhOH Red (500ul/ml, filter sterilized), 0.1 X Vt
   - Rat tail collagen (Volume calculated as above), Vc
   - dH₂O (filter sterilized) to bring up to the Total volume, Vt – Vc – NaOH – PBS Volume
   Add 1M NaOH (0.0231 X Vc). May need to add more 1M NaOH until solution turns pink/purple. This is important to ensure correct polymerization of the gel.
   Incubate the coated plates at 37°C for 10-20 min before seeding cells.
   • Alternative: 0.1% Gelatin Solution (EMD Millipore). Add 10 ml per 10 cm tissue culture dish. Leave the gelatin solution on the plate for at least 30 minutes at room temperature with dish lids on. Discard the solution after the coating process and the plate is ready to use.
10. Centrifuge at 1200 rpm, 5 minutes to pellet the tissue.
11. Carefully aspirate the supernatant. Wash the tissue with PBS.
12. Add 2ml trypsin-EDTA (0.25%, 1X) (Gibco) and incubate at 37 °C for 3 minutes.
13. Wash with PBS and remove supernatant by aspiration.
14. Resuspend the tissue in PDEC medium (10 ml per pancreas) and plate in a 10 cm collagen/gelatin-coated tissue culture dish. Culture in humidified incubator at 37°C, 5% CO₂.
15. After 2 days, the supernatant cellular fraction will be harvested followed by washing with PBS.
16. Add 2ml trypsin-EDTA (0.25%, 1X) and incubate at 37 °C for 3 minutes followed by washing with PBS.
17. Prepare agarose-coated plate: make 1.6% agarose solution in PBS and mix it with PDEC medium at 1:1 ratio. For 6-well plate, add 2 ml of agarose/PDEC medium solution per well and wait it until solidified.
18. Resuspend cells with 1.25 ml PDEC-Matrigel (Corning) (1.5:1 ratio) solution per well of 6-well plate. Consider 3 wells per pancreas. Incubate cells at 37°C, 5% CO₂.
19. Once spheroids form, passage spheroids every 7-8 days depends on health and confluence.
     

Passaging Mouse Pancreatic Epithelial Cells in Spheroid Culture

1. Melt PDEC-Matrigel gel layer with Collagenase IV/Dispase II solution (see above). Add 1.5 ml Collagenase IV/Dispase II solution per well of 6-well plate and incubate at 37 °C for 1-1.5 hours.
2. Collect melted solution in a falcon tube followed by washing with PBS. Pellet cells by centrifugation at 1200 rpm for 5 minutes and discard supernatant.
3. Trypsinize cells for 5 minutes with 1 ml trypsin/EDTA (2.5%, 1X). Wash with PBS and pellet cells by centrifugation at 1200 rpm for 5 minutes followed by discarding supernatant.
4. Prepare agarose-coated plate (see above).
5. Resuspend cells with 1.25 ml PDEC-Matrigel (Corning) (1.5:1 ratio) solution per well of 6-well plate. Keep subculture ratio between 1:2-1:3.
6. Passage spheroid every 7-8 days depends on health and confluence.