Abstract: 【Abstract】 Objective To establish a culture method for primary human nail matrix cells in serum-free media. Methods Nail matrix tissues were collected from 9 patients, who received nail or toe amputation and nail bed repair in Peking University Shenzhen Hospital between January 2016 and December 2016, and cultured in the serum-free DEME/F-12 media at a 37℃ incubator with an atmosphere of 5% CO2 in air for 2 - 3 days. Then, primary human nail matrix cells were cultured in keratinocyte serum-free media （CnT-07）, and the morphology of human nail matrix cells was observed by microscopy during the culture process. Immunofluorescence cytochemistry with anti-keratin 5 （K5） and K10 was performed to identify the acquired cells, and flow cytometry to analyze the cell purity. Results After 2 or 3 days of the culture, some cells began to crawl out from the tissue. On day 10, large cell masses were formed, some cells were morphologically similar to epithelioid cells arranged in a paving stone-like pattern, and some were flat giving a spindle-shaped or star-shaped appearance. Immunofluorescence cytochemistry showed that some cells could express both K5 and K10, which proved the existence of nail matrix cells, and 37.6% of the cells expressed K10. Conclusion Human primary nail matrix cells could be successfully cultured by using the tissue culture method with serum-free culture media, and the nail matrix cells cultured in vitro can express both K5 and K10.

Key words: Nails, Primary cell culture, Culture media, serum?free, Keratin?5, Keratin?10, Nail matrix cell