Separation of organoids into single cells

(Adapted from Genes & Dev. 2002 16: 693-706

  1. Thaw a vial of collagenased pellet of human breast organoids and add 1 ml of F12 media. (If vial is enriched for epithelial cells- go to step 2. If heavy stromal contamination, plate the thawed suspension in a 10cm plate with DMEM/F12 + 5% serum for 1-2 hours to allow stromal cells to attach). Collect supernatant and spin down. Resuspend in 2 mls of F12 media and proceed to step 2.
  2. Pass the suspension through an 18G need ~ 8times into a 15ml conical.
  3. Rinse syringe with a few mls of F12 media.
  4. Spin cells at 1600 rpm for 5 min and (only if needed, resuspend pellet in 1 ml of RBC lysis buffer for 1 min- wash with 10 mls of PBS and spin)
  5. Resuspend pellet in 2 mls of 0.25% Trypsin/EDTA for 2 min at 37°C.
  6. Using a P1000, break up DNA aggregates
  7. Add 5 mls of serum free F12 I/E/H with 2 mls of 1U/µl DNase. Incubate for 5 min at 37°C
  8. Add ~6mls of DMEM/F12+5%CS to neutralize serum
  9. Filter suspension through a 40µm cell strainer into a 50ml conical. Wash with 10mls of DMEM/F12+5%CS.
  10. Spin cells at 1600 rpm for 5 min and resuspend in 5mls of PBE.
  11. Count cells and plate