Fibroblast Subculturing Protocol

Subculturing, or passaging, is the process of splitting and transferring cells from one culture vessel to another to maintain a healthy, proliferative cell population. Here is a general fibroblast subculturing protocol:

  1. Prepare materials and reagents:
  • Sterile phosphate-buffered saline (PBS)
  • Trypsin-EDTA solution (0.25% trypsin with 0.02% EDTA)
  • Complete growth medium (e.g., Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin)
  • Sterile culture vessels (e.g., T-25 or T-75 flasks)
  • Sterile pipettes and pipette tips
  • Hemocytometer for cell counting (optional)
  1. Pre-warm reagents and medium to 37°C in a water bath or incubator.
  2. Observe the fibroblast culture under an inverted microscope. If the cells are 80-90% confluent, they are ready for subculturing.
  3. Aspirate the culture medium from the flask and discard it.
  4. Wash the cell monolayer gently with sterile PBS to remove residual medium and dead cells. Aspirate and discard the PBS.
  5. Add an appropriate volume of pre-warmed trypsin-EDTA solution to the flask (e.g., 1-2 mL for a T-25 flask or 2-3 mL for a T-75 flask). Incubate the flask at 37°C for 2-5 minutes, or until the cells have detached. Monitor cell detachment under an inverted microscope.
  6. Once the cells have detached, gently tap the flask to ensure complete cell detachment. Add an equal volume of complete growth medium to the flask to neutralize the trypsin.
  7. Transfer the cell suspension to a sterile centrifuge tube. Centrifuge the cells at 200-300 x g for 5 minutes to pellet the cells.
  8. Aspirate and discard the supernatant, being careful not to disturb the cell pellet.
  9. Resuspend the cell pellet in an appropriate volume of fresh, pre-warmed complete growth medium.
  10. Optional: Count the cells using a hemocytometer to determine the cell concentration.
  11. Dilute the cell suspension to the desired seeding density, usually between 5,000 and 10,000 cells/cm². Transfer the appropriate volume of cell suspension to a new sterile culture vessel containing fresh complete growth medium.
  12. Incubate the newly seeded culture vessel at 37°C in a humidified atmosphere with 5% CO₂. Monitor cell growth and passage the cells again when they reach 80-90% confluency.

Note: This is a general protocol for subculturing fibroblasts. Specific cell lines or primary cells may require modifications to this protocol, such as using different media or supplements. It is essential to follow the recommendations provided by the cell supplier or consult relevant literature for optimal growth conditions. Always maintain aseptic technique when working with cell cultures to avoid contamination.