1. Neonatal dermal melanocytes are pigment-producing cells;

2. Skin pigmentation is a multi-dimensional and complex process; Melanin, produced in melanocytes, is packaged in melanocytes, then exocytosis out of the cell and endocytosis by adjacent keratinocytes.

3. Many genes play a role in controlling pigmentation and melanin production at different levels.

4. These genetic mutations are characteristic of a variety of skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyperpigmentation/hypopigmentation.

5. In addition, external factors secreted by other suhhounding intrinsic cells also regulate the expression of melanin in melanocytes.

6. Human primary cells can serve as useful models to elucidate the biology of melanocytes and their toxicity to skin agents. However, primary cells have their limitations, such as donor variability and limited lifespan. Therefore, researchers need a more robust model system of human melanocytes to study skin pigmentation and the factors that affect it.

Human immortalized neonatal skin melanin cell line: CRL-4064 developed according to customer requirements

1. A human immortalized neonatal skin melanin cell line was established by inserting overexpressed human telomerase reverse transcriptase (hTERT) protein into primary neonatal skin melanocytes (ATCC?PCS-200-012?).

2. This newly established cell line can be used as a complement to the cell model of human immortalized dermal melanin cell line (ATCC?CRL-4059?) in vitro toxicity studies and pigmentation regulation.

3. And the newborn cells came from an Asian donor.

黑色素細胞標記物表達

Figure 1. Immortalized melanocytes uniformly expressed melanocyte specific marker TRP1, but did not express fibroblast specific marker TE7. hTERT melanocytes were immobilized with 4% paraformaldehyde and then infiltrated with 0.2%Triton x-100, 0.1% Tween-20, and 10% normal goat serum solution on PBS. The cells were then stained in a PBS solution containing 0.05% tween-20 and 2%NGS in a 1:400 murine-human primary antibody. The cells were then washed and stained with 1:400 diluted Alexa fluor 488 bound goat anti-mouse IgG. Finally, the cells were re-stained with 1:500 Hoechst nuclear staining diluted with PBS and observed with Nikon Ti-eclipse imaging inverted microscope and 20x objective lens. (Scale is 100 microns)

Cell morphology

Figure 2. The morphology of hTERT immortalized newborn melanocytes is similar to that of typical melanocytes. The fusion images of the adherent melanocytes were captured by high power and low power inverted microscopes under 10x objective. (Scale is 82 microns)

Q1. How is hTERT neonatal melanin cell line produced?

A: CRL-4064 is produced by retrovirus transduction. Virus particles were produced by transfection of BABE-puro-hTERT plasmid into Phoenix AMPHO (CRL-3213) packaging cells. After the primary cells were infected with the virus, they were subjected to antibiotic pressure selection, and positive single-cell clones were selected.

Q2. How is immortalization determined?

A: From two aspects, the longevity test: the cell line did not decrease in growth rate after doubling in 35 populations, nor did telomerase decrease, and activity was detected by TRAP (telomerase repeat amplification method). These cells showed high (≥ 8 repeat DNA units) telomerase activity compared to CRL-1573-positive controls.

Q3. What is the recommended medium for hTERT newborn melanocytes?

A: The cell line was grown in dermal cell base medium (PCS-200-030) with adult melanocyte growth kit (PCS-200-042) and 0.5 μg/ml purinomycin (to maintain hTERT expression).

Q4: What are the main uses of the CRL-4064 cell line?

A: CRL-4064 is intended to replace primary cells for toxicological or drug screening applications in response to the short life span of primary cells in culture. The cells could also be used in gene regulation studies.

Q5、What is the difference between Q5, primary melanocytes and hTERT melanocytes?

A: hTERT melanocytes continued to divide beyond the typical Hayflick limit of ~15 PDL, and they did not even show growth ahhest at high passage times. ATCC provided two hTERT immortalized melanin cell lines: CRL-4064 for neonatal donors and CRL-4059 for adult donors.