HaCaT cell culture

Protocol for Culturing HaCaT Cells

Materials and Reagents

• HaCaT cells
• DMEM containing 4.5 g/L glucose, sodium pyruvate, and GlutaMAX
• Fetal bovine serum (FBS)
• Penicillin-streptomycin
• Phosphate-buffered saline (PBS)
• Accutase
• DMSO
• Cell counter or hemocytometer

Complete Culture Medium

Prepare DMEM containing 4.5 g/L glucose, sodium pyruvate, and GlutaMAX, supplemented with 10% FBS and 1% penicillin-streptomycin. Warm the required volume before use and handle all reagents aseptically.

Culture Conditions

• Incubate at 37 °C in a humidified atmosphere containing 5% CO₂.
• Maintain cells as an adherent monolayer.
• Replace culture medium every second day or as required.
• Passage cells at approximately 70–80% confluency; avoid prolonged over-confluence.

Thawing and Initial Recovery

1. Rapidly thaw one cryovial and transfer the cells into complete culture medium.
2. Seed into an appropriately sized flask and incubate overnight.
3. Replace the medium the following day to remove residual DMSO and non-adherent material.
4. Monitor attachment, morphology, confluency, and contamination.

Routine Maintenance and Passaging

1. At 70–80% confluency, aspirate the medium and wash once with PBS.
2. Add enough Accutase to cover the monolayer and incubate for approximately 7 minutes, or until cells detach.
3. Add an equal volume of complete medium and disperse to a single-cell suspension.
4. Centrifuge at 300 × g for 5 minutes, remove the supernatant, and resuspend in fresh complete medium.
5. Count the cells and seed at the required density into fresh culture vessels.

Cryopreservation

1. Harvest and count a healthy, actively growing culture.
2. Centrifuge at 300 × g for 5 minutes and remove the supernatant.
3. Resuspend at 1 × 10⁶ cells/mL in chilled freezing medium; the source formulation uses FBS containing 20% DMSO.
4. Dispense 1 mL per cryovial and freeze at approximately −1 °C per minute to −80 °C.
5. Transfer to long-term cryogenic storage after the initial overnight freeze.

Quality and Safety Notes

• Use aseptic technique and follow local biosafety procedures.
• Regularly confirm cell identity, mycoplasma status, morphology, and growth.
• Use consistent passage ranges and adapt volumes and seeding densities to the culture vessel and experiment.