Primary Epidermal Keratinocytes; Normal, Human, Adult (HEKa) (ATCC® PCS-200-011)

Organism: Homo sapiens, human  /  Tissue: skin (epidermis)  /  Cell Type: keratinocyte

Permits and Restrictions

View Permits

Organism Homo sapiens, human
Tissue skin (epidermis)
Cell Type keratinocyte
Morphology Cobblestone appearance; cells are rounded, not flat; cells display a high mitotic index; at near 80% confluence, the cells will be associated with each other in colonies.
Growth Properties adherent
Biosafety Level 1

[These primary cells are not known to harbor an agent recognized to cause disease in healthy adult humans. Handle as a potentially biohazardous material under at least Biosafety Level 1 containment. Cells derived from primate lymphoid tissue may fall under the regulations of 29 CFR 1910.1030 Bloodborne Pathogens.  

ATCC recommends that appropriate safety procedures be used when handling all primary cells and cell lines, especially those derived from human or other primate material. Detailed discussions of laboratory safety procedures are provided in Laboratory Safety: Principles and Practice, 2nd ed. (ASM Press, Washington, DC) (Fleming et al., 1995) and Caputo, J.L. Biosafety procedures in cell culture. (1988) J. Tissue Culture Methods 11:223.

Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online at]

Human Material Precaution 
All tissues used for isolation are obtained under informed consent and conform to HIPAA standards to protect the privacy of the donor’s personal health information. It is best to use caution when handling any human cells. We recommend that all human cells be accorded the same level of biosafety consideration as cells known to carry HIV. With infectious virus assays or viral antigen assays, even a negative test result may leave open the possible existence of a latent viral genome.

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease normal
Age adult
Gender lot-specific
Ethnicity lot-specific
Toxicology, wound repair, skin cancer, response to UV radiation, psoriasis, eczema, viral infection, gene delivery systems, cellular differentiation, cosmetics research/testing
Product Format frozen 1 mL
Storage Conditions -130°C or below
A complete solution to propagate keratinocytes isolated from human skin. The serum-free media is formulated to inhibit fibroblast growth, and the low calcium concentration (60 µM) slows differentiation. No feeder layers, extracellular matrix proteins or other substrates are required.
Complete Growth Medium
  1. Obtain one Keratinocyte Growth Kit from the freezer; make sure that the caps of all components are tight.
  2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.
  3. Obtain one bottle of Dermal Cell Basal Medium (485 mL) from cold storage.
  4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
  5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component, as indicated in Table 1, to the bottle of basal medium using a separate sterile pipette for each transfer.
  6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
  7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.

   Table 1. Keratinocyte Growth Kit Components 





Final Concentration


Bovine Pituitary Extract (BPE)


2.0 mL




rh TGF-a


0.5 mL


0.5 ng/mL




15.0 mL


6 mM


Hydrocortisone Hemisuccinate


0.5 mL


100 ng/mL


rh Insulin


0.5 mL


5 mg/ml




0.5 mL


1.0 mM




0.5 mL


5 mg/ml



Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components (GA solution or PSA solution) to be added to the complete growth media is summarized in Table 2.


Table 2. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional)





Final Concentration


Gentamicin-Amphotericin B Solution


0.5 mL


Gentamicin: 10 µg/mL

Amphotericin B: 0.25 µg/mL


Penicillin-Streptomycin-Amphotericin B Solution


0.5 mL


Penicillin: 10 Units/mL

Streptomycin: 10 µg/mL

Amphotericin B: 25 ng/mL


Phenol Red


0.5 mL


33 µM




  1. Passage normal keratinocytes when the culture has reached approximately 70% to 80% confluence.
  2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete growth medium to 37°C prior to use with the cells.
  3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
  4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
  5. Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm2) to each flask.
  6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.
  7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 6 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.
    If cells are difficult to detach, incubate each flask containing cells and the trypsin-EDTA solution at 37°C to facilitate dispersal.
  8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
  9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the culture flask.
  10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that might have been left behind.
  11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-dissociated cells.
  12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
  13. Centrifuge the cells at 150 x g for 3 to 5 minutes.
  14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
  15. Count the cells and seed new culture flasks at a density of 2,500 to 5,000 cells per cm2.
  16. Place newly seeded flasks in a 37°C, 5% CO2 incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance for guidelines on feeding.
Volume 1 mL
Cells per Vial One vial contains a minimum of 5 x 105  viable cells.
Sterility Tests
Bacteria and Yeast: Negative
Mycoplasma: Negative
Viral Testing
Hepatitis B: Negative
Hepatitis C: Negative
HIV: Negative
Viability ≥ 70% when thawed from cryopreservation
Population Doubling Capacity ≥ 15 in complete growth medium
C of A
Certificate of Analysis
Certificate of Analysis
Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation

Kalyan Kumar G, et al. Embelin Reduces Cutaneous TNF-a Level and Ameliorates Skin Edema in Acute and Chronic Model of Skin Inflammation in Mice. Eur J Pharmacol 662(1-3):63-9, 2011. PubMed: 21549694

Madigan MC, et al. Xanthine Oxidoreductase Function Contributes to Normal Wound Healing. Mol Med 21(1): 313-322, 2015. PubMed: 25879627

Lim PY, et al. Keratinocytes Are Cell Targets of West Nile Virus In Vivo. J Virol 85(10):5197-201, 2011. PubMed: 21367890

Rezusta A, et al. In Vitro Fungicidal Photodynamic Effect of Hypericin on Candida Species. Photochem Photobiol 88(3):613-9, 2012. PubMed: 22128758

Sun Z, et al. miR-21 Increases the Programmed Cell Death 4 Gene-Regulated Cell Proliferation in Head and Neck Squamous Carcinoma Cell Lines. Oncol Rep 32(5):2283-9, 2014. PubMed: 25175929

Catanzano O, et al. Advanced Multi-targeted Composite Biomaterial Dressing for Pain and Infection Control in Chronic Leg Ulcers. Carbohydr Polym 172:40-48, 2017. PubMed: 28606546

Zhao Y, et al. Exposure to Titanium Dioxide Nanoparticles Induces Autophagy in Primary Human Keratinocytes. Small 9(3):387-92, 2013. PubMed: 23090781

Mi G, Webster T. Skin Permeable Peptide Amphiphiles as Novel Anti-Aging and Anti-Microbial Agents. Front Bioeng Biotechnol 2016. Conference Abstract: 10th World Biomaterials Congress. DOI: 10.3389/conf.FBIOE.2016.01.02846

Deng Y, Ma W. Metformin Inhibits HaCaT Cell Viability Via the miR-21/PTEN/Akt Signaling Pathway. Mol Med Rep 17(3):4062-4066, 2018. PubMed: 29286158

Ahmed A, Boateng J. Calcium Alginate-Based Antimicrobial Film Dressings for Potential Healing of Infected Foot Ulcers. Ther Deliv 9(3):185-204, 2018. PubMed: 29424287