Primary Human Melanocytes and Growth Media

These products are available in the US, Canada, and select European countries.

• Primary Human Melanocyte Culture System
• Quality Control for Human Melanocytes and Growth Media
• Melanocyte Culture Materials

Primary Human Melanocyte Culture System

Epidermal melanocytes, located in the stratum basale with extensions to the suprabasal layers, make up 5-10% of the cells in the epidermis. Epidermal melanocytes produce the protein melanin, which is responsible for pigmentation of the skin, hair and eyes, in addition to a UV-shielding effect on adjacent cells.

PromoCell Normal Human Epidermal Melanocytes are generated from donors with varied skin pigmentation (light, moderate, and dark). Detailed pigmentation information is available upon request. The isolated cells originate from juvenile foreskin or adult skin from the face, breast, abdomen, thighs, and other sites. The media used is either the serum-free, Melanocyte Growth Medium with PMA (Phorbol Myristate Acetate), or the unique serum-free and PMA-free Melanocyte Growth Medium M2. All Normal Human Epidermal Melanocytes are cryopreserved at passage 2 (P2) shortly after isolation, using a proprietary, serum-free freezing medium, Cryo-SFM. Each thawed cryovial contains at least 500,000 viable cells. Human Melanocytes are also available as proliferating cell cultures made from cryopreserved cells that have been thawed and cultured for three days.

• Cultivation Protocol for Primary Human Melanocytes

Melanocyte Growth Media was developed for the culture of primary human melanocytes and is available in two distinct formulations:

• Melanocyte Growth Medium with PMA (Phorbol Myristate Acetate) is optimized for growing primary human cells, but can also be used for equine, porcine, mouse, and rat melanocytes.
• Melanocyte Growth Medium M2, is a unique serum-free and PMA-free medium that is formulated for growing human melanocytes in vitro. The medium is also suitable for human pigment epithelial cells, neuronal cells, and melanoma cells. It is optimized for growing primary human cells, but can also be used for equine, porcine, mouse, and rat melanocytes. In addition to cultivation, we recommend using melanocytes isolated in Melanocyte Growth Medium M2 to avoid the tumor-promoting
  activity of PMA.

The Melanocyte Growth Medium containing PMA is available in two formats: Medium (ready-to-use) and Medium Kit. The Medium (ready-to-use) option includes a 500 mL bottle of Basal Medium and one vial of SupplementMix. The Medium Kit includes a 500 mL bottle of Basal Medium and the SupplementPack, which is a set of individual vials with pre-measured supplements that affords the user full control over the complete media formulation. Adding the SupplementMix or the SupplementPack to the Basal Medium makes the complete Melanocyte Growth Medium. The Melanocyte Basal Medium (with or without phenol red), SupplementMix, and SupplementPack can also be purchased separately.

The Melanocyte Growth Medium M2 (PMA-free) is only available as Medium (ready-to-use), which includes a 500 mL bottle of Basal Medium and one vial of SupplementMix. Adding the SupplementMix to the Basal Medium generates the complete Melanocyte Growth Medium M2. The Melanocyte Basal Medium M2 (with or without phenol red) and the SupplementMix can also be purchased separately.

Table 1. Recommended Melanocyte Cell Types for Melanocyte Growth Medium (with PMA)

Melanocyte Growth Medium (with PMA)	Melanocyte Growth Medium M2 (PMA-free)
Normal Human Epidermal Melanocytes (NHEM), juvenile foreskin	Normal Human Epidermal Melanocytes (NHEM), juvenile foreskin, cultured in M2 Medium Normal Human Epidermal Melanocytes (NHEM), adult donor, cultured in M2 Medium

Human Melanocyte Growth Media Supplementation Details

The Melanocyte Growth Media includes all of the necessary components for the optimal growth of human melanocytes. Melanocyte Growth Media does not contain antibiotics or antimycotics and is formulated for use in an incubator with 5% atmospheric CO₂.

Table 2. Final supplement concentrations in Melanocyte Growth Medium (after addition)

Component	Melanocyte Growth Medium	Melanocyte Growth Medium M2
Bovine Pituitary Extract	0.004 mL / mL	Proprietary serum-free, PMA-free formulation
Basic Fibroblast Growth Factor (recombinant human)	1 ng / mL
Insulin (recombinant human)	5 μg / mL
Hydrocortisone	0.5 μg / mL
Phorbol Myristate Acetate	10 ng / mL

Preparing Supplemented Media for Human Melanocyte Culture

• Thaw the Melanocyte SupplementMix or SupplementPack at 15 – 25 °C.
• Aseptically mix the supplement solutions by pipetting up and down gently.
• Transfer the entire contents of each supplement to the Melanocyte Basal Medium.
• Close the bottle and swirl gently to achieve a homogeneous mixture.

Storage and Stability of Melanocyte Products

• Upon arrival, immediately store the Melanocyte Basal Medium in the dark at 4 – 8 °C; do not freeze. Store the Melanocyte Growth Medium SupplementMix or SupplementPack at -20 °C.
• If stored as directed, the products are stable until the expiration date stated on the label.
• Once supplements are added to the Melanocyte Basal Medium, the shelf life of the complete medium is six weeks at 4 – 8 °C. Do not freeze the complete medium.
• To use, aliquot and prewarm just the volume needed of the complete Melanocyte Growth Medium and keep the remaining medium refrigerated at 4 – 8 °C.
• Upon arrival, store cryopreserved melanocyte cells in liquid nitrogen or seed directly.
• Proliferating melanocyte cells must be processed immediately.

Quality Control for Human Melanocytes and Growth Media

Normal Human Epidermal Melanocytes

The following rigid quality control tests are performed for each lot of Human Epidermal Melanocytes:

• Cell morphology, adherence rate, and cell viability testing
• Immunohistochemical testing for the cell-type specific marker Mel-5
• Growth performance testing through multiple passages up to 15 population doublings (PD) under culture conditions without antibiotics and antimycotics
• Testing for the absence of HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2 and microbial contaminants (fungi, bacteria, and mycoplasma)
• A detailed certificate of analysis (CoA) for each lot is available for download.

Normal Human Epidermal Melanocytes Specifications

Table 3. *catalog numbers in this table are for media in ready-to-use packaging

Product	Recommended Culture Medium*	Plating Density	Passage after Thawing	Marker	Population Doublings
Normal Human Epidermal Melanocytes (NHEM), juvenile foreskin	C-24010	5,000 - 10,000 cells per cm²	P2	Mel-5+	> 15
Normal Human Epidermal Melanocytes (NHEM), juvenile foreskin, cultured in M2 Medium	C-24300	5,000 - 10,000 cells per cm²	P2	Mel-5+	> 15
Normal Human Epidermal Melanocytes (NHEM), adult donor, cultured in M2 Medium	C-24300	5,000 - 10,000 cells per cm²	P2	Mel-5+	> 15

Quality Control for Epidermal Melanocyte Growth Media

All lots of Melanocyte Growth Media are subjected to comprehensive quality control testing. These tests check for growth-promoting activity, adherence rate, and typical cell morphology using cultured primary human melanocytes. Approved in-house lots of Melanocyte Growth Media are used as a reference. Additionally, microbial testing ensures that all lots of Epidermal Melanocyte Growth Media are free of microbial contaminants (fungi, bacteria, mycoplasma).

Intended Use for Human Melanocytes and Media

Human Melanocytes and Growth Media are intended for in vitro use only and not for diagnostic or therapeutic procedures. For safety information please see appropriate MSDS.

Note: PromoCell^® media have low or zero serum content and are not suitable for trypsin neutralization (when subculturing the cells, for example). Instead, we recommend using the DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.