Fibroblast Cell Culture Protocol

Fibroblast Subculturing Protocol

In cell culture, fibroblasts should be grown in 90% RPMI 1640 medium with 10% FBS added. A media alternative includes alpha-MEM and Dulbecco’s modified MEM (i.e. DMEM).

  1. Rinse the flask with sterile 1x PBS to remove all complete medium; any remaining media will interfere with detachment of cells
  2. To the flask, add enough trypsin-EDTA to cover the bottom of the flask; observe the flask for cell layer detachment under an inverted microscope.  Incubate at 37°C with 5% CO2 for 4 to 7 minutes. Cells should round up and become dislodged; encourage detachment by very lightly tapping the side of flask.
  3. Neutralize the trypsin-EDTA activity by adding complete media at a volume of 4x the volume of trypsin-EDTA added to stop the reaction; centrifuge the cells and remove the supernatant.
  4. Gently pipette to resuspend the cells and determine cell density (i.e. hemocytometer or cell counter)
  5. Reseed flasks to a total volume of 15-30 mL such that the split ratio is 1:5 to 1:10. After 2-3 days the monolayer will be confluent; split 2-3 times a week. Initially, cells should be seeded at about 1-2 x 106 cells/25 cm2. If splitting less frequently, replace medium every 3-4 days.
  6. Incubate at 37°C in 5% CO2.

Troubleshooting Subculture Procedures

If fibroblast cells are difficult to detach from the flask:

  • Cells are too confluent; cell-to-cell junctions are extremely tight and preventing dissociation agents from reaching the cell interface; this problem is resolved by ensuring the subculturing process occurs before the cells become confluent
  • The dissociation agent is at a low concentration and a higher concentration must be used; place the flask containing the dissociation agent in the incubator while waiting for detachment to increase enzymatic activity
  • Ensure the flask was washed completely with sterile 1x PBS prior to addition of the dissociation agent; if necessary, use two PBS washes to remove media

Cells form clumps after detachment:

  • Centrifugation was too harsh; do not spin cells harder than 100 x g for 5 minutes to pellet
  • Place the flask or vial on ice after resuspending; this will decrease cell aggregation

Cell viability is low after passaging cells:

  • Reduce harsh pipetting during cell collection
  • Only leave dissociation agent on cells as necessary to detach cells


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Fibroblast cell line culturing protocol – Fibroblast Cell Culture FAQ

siRNA in vivo delivery – InVivoTransfection Resource

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Research Studies:

  • A study of cytomegalovirus infection in fibroblasts: This research utilized immortalized human fibroblast cell lines to study human cytomegalovirus infection. It determined that the immortalized cells were similar to the original human cells in their response to the infection, paving the way for future study of cytomegalovirus infection. [LINK]
  • Utilizing human fibroblasts as a substrate for corneal epithelium regeneration: In order to avoid xeno-component use in the generation of human tissue for corneal reconstruction, this study screened five human fibroblast cell lines for use as a feeder layer to cultivate corneal cells. The regenerated epithelial cells were evaluated by morphology, immunostaining, and gene expression. It was found that the commercial human fibroblast cell lines were able to support corneal cell regeneration as well as the previously-used murine cell lines. [LINK]