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Primary Epidermal Melanocytes; Normal, Human, Adult (HEMa)

PCS-200-013

ATCC® Normal Human Primary Epidermal Melanocytes from Adult donors, when grown in Dermal Cell Basal Medium supplemented with Adult 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. ATCC® Primary Cell Solutions™ cells, media, supplements and reagents are quality tested together to guarantee optimum performance and reliability.
Product category
Human cells
Product type
Primary cell
Organism
Homo sapiens, human
Cell type
melanocyte
Morphology
Stellar, multipolar or dendritic apperance; needle-like
Tissue
Skin
Disease
Normal
Applications
3D cell culture
Toxicology
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon BSL 1

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

All tissues used for isolation are obtained under informed consent and conform to HIPAA regulations to protect the privacy of the donor’s Personally Identifiable 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 Human immunodeficiency virus (HIV) and other bloodborne pathogens. With infectious virus assays or viral antigen assays, even a negative test result may not exclude the possibility of the existence of a latent viral genome or infectious viral particles below the lower limit of detection of that assay.

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. Handle as a potentially biohazardous material using universal precautions. Cells derived from primate lymphoid tissue may fall under the regulations of 29 CFR 1910.1030 Bloodborne Pathogens.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Dermal Cell Basal Medium

PCS-200-030

Price: $55.00 ea

Adult Melanocyte Growth Kit

PCS-200-042

Price: $127.00 ea

Dulbecco's Phosphate Buffered Saline (D-PBS), 1X

30-2200

Price: $16.00 ea

Trypsin-EDTA for Primary Cells

PCS-999-003

Price: $28.00 ea

Trypsin Neutralizing Solution

PCS-999-004

Price: $28.00 ea

Penicillin-Streptomycin-Amphotericin B Solution

PCS-999-002

Price: $28.00 ea

Phenol Red

PCS-999-001

Price: $28.00 ea

Detailed product information

General

Specific 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)

Characteristics

Cells per vial
≥ 5.0 x 105
Volume
1.0 mL
Growth properties
Adherent
Age
lot-specific
Ethnicity
Lot-specific
Gender
Lot-specific
Comments
ATCC® Normal Human Primary Epidermal Melanocytes (HEMa) from from adult tissue, when grown in Dermal Cell Basal Media supplemented with Adult Melanocyte Growth Kit components, provide an ideal cell system to propagate adult 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.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete 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.

Adult Melanocyte Growth Kit Components:
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, 840 µL, 1.5 mM
Peptide Growth Factor, 1.0 mL, Proprietary formulation
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 below.

Addition of Antimicrobials/Antimycotics and Phenol Red (Optional):
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.
Reagents for subculture
  1. D-PBS (ATCC 30-2200)
  2. Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) containing 0.05% Trypsin and 0.02% EDTA. Note: Do not use other trypsin-EDTA concentrations with ATCC PCS-200-013.
  3. Trypsin Neutralizing Solution (ATCC PCS-999-004)
Required media
One bottle of Dermal Cell Basal Medium (ATCC PCS-200-030) plus one Adult Melanocyte Growth Kit (ATCC PCS-200-042) that contains the following components: L-glutamine, recombinant human (rh) insulin, ascorbic acid, epinephrine, calcium chloride, a proprietary peptide growth factor and M8 Supplement (a proprietary formulation containing FBS and other factors).
Optional media supplements
  1. Gentamicin-Amphotericin B Solution (ATCC PCS-999-025)
  2. Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002)
  3. Phenol Red (ATCC PCS-999-001)
Handling procedure
  1. Refer to the batch specific information for the total number of viable cells recovered from this lot of ATCC PCS-200-013.
  2. Using the total number of viable cells, determine how much surface area can be inoculated to achieve an initial seeding density of 5,000 cells per cm2.
  3. Prepare the desired combination of flasks. Add 5 mL of complete growth medium per 25 cm2of surface area. Place the flasks in a 37°C, 5% CO2, humidified incubator and allow the media to pre-equilibrate to temperature and pH for 30 minutes prior to adding cells.
  4. While the culture flasks equilibrate, remove one vial of ATCC PCS-200-013 from storage and thaw the cells by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes).
  5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point onward should be carried out under strict aseptic conditions.
  6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of flasks to be seeded) - – 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not centrifuge.
  7. Transfer 1.0 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times, then cap and gently rock each flask to evenly distribute the cells.
  8. Place the seeded culture flasks in the incubator at 37°C, 5% CO2 atmosphere. Incubate for at least 24 hours before processing the cells further.
Subculturing procedure
  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. 
Culture maintenance
  1. Before beginning, pre-warm complete growth media in a 37°C water bath. This will take between 10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less), warm only the volume needed in a sterile conical tube. Avoid warming complete growth media multiple times.
  2. 24 to 36 hours after seeding, remove the cells from the incubator and view each flask under the microscope to determine percent cellular confluence.
  3. Carefully remove the spent media without disturbing the monolayer.
  4. Add 5 mL of fresh, pre-warmed complete growth medium per 25 cm2 of surface area and return the flasks to the incubator.
  5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have reached 80% to 90% confluence, and are actively proliferating (many mitotic figures are visible), it is time to subculture. Melanocytes are not contact inhibited; however, they will proliferate best if the cells are passaged prior to 100% confluence. Melanocytes from lightly pigmented tissue should reach 80% confluence in 7 to 9 days. 

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Human immunodeficiency virus 1 (HIV-1): Not detected
Hepatitis C virus (HCV): Not detected
Hepatitis B virus (HBV): Not detected
Population doubling capacity
≥ 15 in complete growth medium
Viability
≥ 70% when thawed from cryopreservation
Specific staining
Positive for L-DOPA Oxidase activity.
Positive for Mel5, negative for Pan-CK, negative for TE-7

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Camilio KA, et al. Complete Regression and Systemic Protective Immune Responses Obtained in B16 Melanomas after Treatment with LTX-315. Cancer Immunol Immunother 63.6(63):601-13, 2014. PubMed: 24676901

Miniati A, et al. Stimulated Human Melanocytes Express and Release Interleukin-8, Which Is Inhibited by Luteolin: Relevance to Early Vitiligo. Clin Exp Dermatol 39.1(39):54-7, 2014. PubMed: 23782102

Ding Z, et al. Loss of MiR-664 Expression Enhances Cutaneous Malignant Melanoma Proliferation by Upregulating PLP2. Medicine (Baltimore) 94.33(94):e1327, 2015. PubMed: 26287415

Lipiec E, et al. Monitoring UVR Induced Damage in Single Cells and Isolated Nuclei Using SR-FTIR Microspectroscopy and 3D Confocal Raman Imaging. Analyst 139.17(139):4200-9, 2014. PubMed: 24995477

Tian, F et al. miR-33a Is Downregulated in Melanoma Cells and Modulates Cell Proliferation by Targeting Pctaire1. Oncol Lett 4(11):2741-46, 2016. PubMed: 27073545

View All Curated Citations for this Product

Frequently Asked Questions

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