Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices

Abstract

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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Fig. 1: Workflow from freshly isolated keratinocyte culture to animal transplantation.
Fig. 2: Flow diagram of fibrin mat preparation.
Fig. 3: Representative images of human epidermal keratinocytes cultured on the LN system.
Fig. 4: Cross-section of cultured human epidermal keratinocyte monolayer on fibrin coated with LN-511 or LN-421.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

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.

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M.S.T designed the protocol and all experimental procedures, wrote the article and prepared figures. A.W.C.C. and K.T. are both project co-leaders and assisted with the preparation of the article.

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Correspondence to Alvin Wen Choong Chua or Karl Tryggvason.

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Competing interests

K.T. is a shareholder of BioLamina.

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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.

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Tjin, M. S. et al. Nat. Commun. 9, 4432 (2018): https://doi.org/10.1038/s41467-018-06934-3

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Antibodies used in the study

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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

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