Xeno-free expansion of adult keratinocytes for clinical application: the use of human-derived feeder cells and serum
Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green’s methods; however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f; a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.
KeywordsAdult keratinocytes Skin tissue engineering CEA Burns IL-8 (CXCL8) CD49f (α6 integrin)
The authors would like to acknowledge Dr. Stephen Goldie for a critical review of the manuscript and the Alfred Foundation for financial support. Patients and staff of Linacre and Western Private Hospitals, the Alfred and Monash Health are also acknowledged for their contribution in donating discarded skin.
Compliance with ethical standards
Conflict of interest
The authors have no conflict of interest to declare.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committees and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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