Human Epidermal Keratinocytes

Normal Human Epidermal Keratinocytes (NHEK) are primary cells isolated from adult skin. They are used to build reconstructed epidermis for animal-free skin irritation testing and to study UV protection, atopic dermatitis and wound healing.

Under organotypic culture conditions, NHEK stratify into a multilayered, differentiated epidermis, the basis of reconstructed human epidermis models. Combined with dermal fibroblasts such as NHDF-Ad, they also form full-thickness skin equivalents for sensitization and wound healing studies.

37°C, 5% CO₂, saturated humidity.

Subculture the cells at about 75 % confluence. Denser cultures undergo irreversible contact inhibition and stop proliferating. Use the subculture reagents recommended on the certificate of analysis.

Post-thaw viability ≥ 70 % viable cells. More than 80 % confluence within the expected recovery period.

1. Thaw the cryovial quickly in a 37 °C water bath, swirling gently. Just before the last ice has melted, move the vial to a laminar flow hood and decontaminate its outer surface.

2. Transfer the cell suspension into a 15 ml centrifuge tube, add 10 ml of keratinocyte growth medium and centrifuge at 250 × g for 5 minutes.

3. Remove the supernatant and resuspend the cell pellet in 10 ml of keratinocyte growth medium. Check cell number and viability.

4. Divide the suspension between two 25 cm² culture flasks (5 ml each). Using two flasks reduces the risk of losing all cells if a passage fails.

5. Incubate at 37 °C, 5 % CO₂ and saturated humidity, with the screw caps opened by half a turn for gas exchange.

6. Change the medium after 24 hours to remove unattached cells, then every 48 to 72 hours, using pre-warmed medium only.

7. Subculture the cells at about 75 % confluence. Denser cultures undergo irreversible contact inhibition and stop proliferating. Use the subculture reagents recommended on the certificate of analysis.

8. Recommended seeding density: more than 6,000 cells/cm².

Mycoplasma contamination is excluded by PCR.

To ensure there is no bacterial, fungal or yeast contamination, cultures are subjected to visual inspection.

For long-term preservation, store vials in liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable for up to one week only.

Cryopreserved primary cells are shipped on dry ice. On receipt, check the cryovial for damage and transfer it without delay to appropriate storage.

At Cytion, we stand behind the quality and viability of our products. We guarantee that our cells will be viable at the start of culture for 60 days after shipment from our facilities. We assure that all materials conform to the specifications described in the Certificate of Analysis and/or Product Sheet.

For full details, please refer to our Warranty Statement.