A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Automated summary

We developed a new chemically defined serum-free medium, Surge SFM, which can support massive expansion of human primary keratinocytes and production of skin substitutes. Compared to conventional serum-containing medium, Surge SFM can more efficiently expand keratinocytes and properly form stratified epidermis. It also retains keratin-19-positive epithelial stem cells and maintains similar gene expression as with the conventional medium. Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium, which can persist for up to six months when grafted onto mice.