Isolation of Primary Fibroblast from Tissue

Materials
  1. Fresh tissue (Cells were also successfully isolated from frozen tissure but with longer incubation time)
  2. Fisher culture dishes
  3. Instruments Kit, sterile
  4. 6-well plates
  5. T25 flask
  6. T75 flask
  7. 80% Ethanol Spray
  8. PBS
  9. Culture medium: DMEM with 20%FBS and 1%PS and 1%AmpB
  10. Trypsin-EDTA solution
  11. Cell culture incubator (37oC, 5% CO2)
  12. Biosafety hood
Procedure
  1. Clean hood with 80% ethanol spray and lay out appropriate dishes.
  2. Clean instruments with 80% ethanol spray.
  3. Put tissue in dish1 and spray it with ethanol. Use enough ethanol to cover tissue. Leave tissure in EtOH for 90 sec.
  4. Wash the tissue in PBS for 90 sec.
  5. Cut the tissue into 3 roughly equal pieces.
  6. Transfer one of those 3 pieces to the EtOH and wash the tissue in ETOH again for 15sec.
  7. Transfer tissue to the 2nd PBS wash.
  8. Cut this piece into even smaller pieces. Each piece should be cut as small as possible.
  9. Place small tissue pieces in 6-well plates and allow drying by placing plates near the back of the hood for a few minites. Make sure tissue doesn't start turning dark brown. (The medium can be added at different time so there is always a proper drying time for the cells to grow.)
  10. When tissue is relatively dry, add 2-3 ml culture medium and place in incubator.
  11. Change medium periodically.
  12. When the cells spread on the plate and grow to subconfluent, trypsinize the cells and transfer to a T25 flask. (Since the fibroblast cells are easy to detach from the plate, collect the fibroblast at the time just start to detach. Do not over trypsinize the cells. This way you can diminish epithelial cells from the fibroblast cells).
  13. Cells are validated by Western blot with fibroblast specific antibody.

 

 
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