In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics.
- Wong CW School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, 6102, Australia.
- LeGrand CF School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, 6102, Australia.
- Kinnear BF School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, 6102, Australia.
- Sobota RM Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, No. 07-48A Proteos, Singapore, 138673, Singapore.
- Ramalingam R Skin Research Institute of Singapore and Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, 06-06 Immunos, Singapore, 138648, Singapore.
- Dye DE School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, 6102, Australia.
- Raghunath M Centre for Cell Biology and Tissue Engineering, Competence Centre for Tissue Engineering and Substance Testing (TEDD), Institute for Chemistry and Biotechnology, ZHAW School of Life Science and Facility Management, Zurich University of Applied Science, Winterthur, Switzerland.
- Lane EB Skin Research Institute of Singapore and Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, 06-06 Immunos, Singapore, 138648, Singapore.
- Coombe DR School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, 6102, Australia. D.Coombe@exchange.curtin.edu.au.
- 2019-12-08
Published in:
- Scientific reports. - 2019
Cell Culture Techniques
Cell Differentiation
Cell Proliferation
Cells, Cultured
Coculture Techniques
Dermis
Extracellular Matrix
Feeder Cells
Fibroblasts
Humans
Keratinocytes
Skin Transplantation
English
The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell's fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.
- Language
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- English
- Open access status
- gold
- Identifiers
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- DOI 10.1038/s41598-019-54793-9
- PMID 31811191
- Persistent URL
- https://roar.hep-bejune.ch/global/documents/127333
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