Cell Culture Information

HeLa cells are adherent cells grown in Eagle’s MEM (EMEM) modified with 10% FBS. Incubate cells at 37°C with 5% CO2. Renew growth medium 2-3 times a week.  HeLa cell doubling time is 24 hours.

Subculture Protocol for HeLa Cells

To subculture cells:

  1. Aspirate medium from the flask
  2. Add 10 mL sterile 1x PBS and rinse cells by pipette; aspirate cells
  3. Add enough volume (1-2 mL) of Trypsin-EDTA to cover the bottom of the flask; observe flask for cell layer detachment with an inverted microscope
  4. After cells detach, neutralize the dissociation agent by adding 4x volume of complete growth medium and gently pipette to resuspend cells
  5. Add an aliquot of the cell suspension to new culture vessels at the correct split ratio
  6. Incubate at 37°C in 5% CO2

A subcultivation ratio of 1:2 to 1:6 is recommended.

HeLa Cell Freezing Procedure

HeLa cells should be frozen in conditioned growth medium supplemented with 5% (v/v) DMSO and stored in the liquid nitrogen vapor phase.

Tips From the Bench


Cell numbers in flasks must be maintained to fall within certain ranges. Too few cells in the flask results in cells that stop growing due to lack of cell contact.  However, too many cells in the flask causes the cells to stop proliferating from lack of available room to grow.  Much like Goldilocks needing the “just right” conditions, cells must be maintained in exponential growth where flask confluency is maintained between 30-85%.

Thawed Aliquot

Freshly thawed cells are fragile due to DMSO in freezing media.  The frozen vials should be thawed in less than 1 minute. Also, centrifugation of fragile cells adds to cell death; do not pellet freshly thawed cells. Alternatively, DMSO in freezing media will be diluted by pipetting the thawed cell aliquot directly to a flask containing warm media.

Passage Number

Cells undergo genotypic variations and genetic drift with high passage numbers. Record passage numbers on flasks and do not allow cells to reach high numbers. High passage numbers will result in slow growing cells that are hard to transfect and varying data sets that cannot be repeated.


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