The basal keratinocyte progenitor cells in cultured epithelial autografts (CEAs) regenerate human epidermis after transplantation, a curative therapy for severe burns and, recently, diseases with epidermal loss, such as junctional epidermolysis bullosa (EB). Although a culturing technique for skin keratinocytes was developed four decades ago, the xenogeneic nature of that conventional CEA culture system restricts its use to the treatment of critical and life-threatening cases, such as severe burns on >30% of total body surface area and EB. In the present protocol, we describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Skin-specific basement membrane proteins called laminins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. This fully human keratinocyte culture system is ‘regulatory friendly’ and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture. Conservatively, a secondary culture from the primary culture can be expanded up to 20-fold within 4–5 d once cells reach confluency.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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We are thankful to Assoc. Prof. Tan Bien Keem at Plastic, Reconstructive & Aesthetic Surgery, Singapore General Hospital for facilitating collection of skin samples. This study was supported by NMRC STaR Award grants (NMRC/STaR/0010/2012 and MOH-000052) to K.T. and an NMRC grant (NMRC/BNIG/2036/2015) awarded to A.W.C.C.
K.T. is a shareholder of BioLamina.
Peer review information Nature Protocols thanks Ellen Van den Bogaard and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key reference(s) using this protocol
Tjin, M. S. et al. Nat. Commun. 9, 4432 (2018): https://doi.org/10.1038/s41467-018-06934-3
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Tjin, M.S., Chua, A.W.C. & Tryggvason, K. Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices. Nat Protoc 15, 694–711 (2020). https://doi.org/10.1038/s41596-019-0270-3