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Primary Epidermal Melanocytes; Normal, Human, Neonatal Item ID: ATCPCS200012

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Organism Homo sapiens, human
Tissue Foreskin, lightly-pigmented
Cell Type Melanocytes
Morphology Stellar, multipolar or dendritic apperance; needle-like
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 http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.]

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 Neonatal
Gender Male
Applications Melanoma, response to UV radiation, psoriasis and other skin diseases, skin trauma (e.g., wound repair, scars, burns), cosmetic research (e.g., skin lightening compounds, skin protecting compounds)
Product Format frozen 1 mL
Storage Conditions -130°C or below
Comments ATCC® Normal Human Primary Epidermal Melanocytes from Neonatal Foreskin, when grown in Dermal Cell Basal Media supplemented with Melanocyte Growth Kit components, provide an ideal cell system to propagate melanocytes in low serum (less than 1.0% FBS) conditions in the absence of cholera toxin and phorbol 12-myristate 13-acetate (PMA). The cells are cryopreserved in the second passage to ensure the highest viability and plating efficiency.
Complete Growth Medium
  1. Obtain one Melanocyte 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.  Table 1. Melanocyte Growth Kit Components 

    Component

    Volume

    Final Concentration

    rh Insulin

    0.5 mL

    5 µg/ml

    Ascorbic Acid

    0.5 mL

    50 µg/ml

    L-Glutamine

    15.0 mL

    6 mM

    Epinephrine

    0.5 mL

    1.0 µM

    Calcium chloride

    80 µl

    0.2 mM

    M8 Supplement

    5 ml

    Proprietary formulation

     

    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)

    Component

    Volume

    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
  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.
Subculturing
  1. Passage normal melanocytes when the culture has reached approximately 80% to 90% 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 two times 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 1 to 3 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface. Note: Melanocytes are sensitive to over-trypsinization.
  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-1: Negative
Viability ≥ 70% when thawed from cryopreservation.
Population Doubling Capacity ≥ 15 in complete growth medium
C of A Certificate of Analysis
Permits

These permits may be required for shipping this product to Australia:

  • DAFF Import Permit formerly known as AQIS Import Permit must be obtained and a copy of the permit must be sent to ATCC in advance of shipment.
Basic Documentation Product Sheet Certificate of Analysis SDS

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