Keratinocytes and Adipose-derived mesenchymal stem cells: The heir and the spare to regenerative cellular therapies for difficult-to-heal skin wounds

Cell-based therapy is considered as Advanced Therapy Medicinal Product, (ATMP), which had increasingly stricter regulations in the last decade. The cells must be produced according to the ‘Guidelines on Good Manufacturing Practice (GMP) specific to Advanced Therapy Medicinal Products’, adopted by the European Medicines Agency (EMA). A fully compliant autologous keratinocyte-based ATMP certified for clinical use remains an unmet challenge in Europe. This necessitates the development of a comprehensive bio-production workflow to tackle key technical bottlenecks along this procedure. On the other hand, adipose-derived mesenchymal stem cells (AD-MSCs) hold promise as an effective alternative to primary keratinocytes in treating difficult-to-heal wounds, particularly for patients with extensive skin wounds. The overall aim of this thesis is to provide a bio-production workflow addressing the challenges associated with developing an autologous keratinocyte-based ATMP. Additionally, the thesis aims to elucidate the molecular and functional mechanisms that modulate the wound healing capabilities of keratinocytes and AD-MSCs. In papers I-III the bio-production procedure for an autologous keratinocyte-based ATMP to treat difficult-to-heal wounds was divided into 3 main stages; keratinocytes extraction, expansion, and transportation. Paper I validated the use of an animal-origin-free enzymatic workflow for the extraction of keratinocytes from the epidermis, compared to the classical workflow containing animal-derived products. Both workflows proved comparable in efficiency in terms of the final cell yield from skin samples, in addition to the purity and functionality of the keratinocytes following cultivation. This report confirms the feasibility of an entirely xeno-free workflow for acquiring GMP-compliant epidermal cells suitable for clinical application without altering key features of keratinocytes. Paper II evaluates an expansion approach for keratinocytes on three culture substrates (1) glass (2) conventional polystyrene (plastic) and (3) animal-derived collagen I ECM matrix. Keratinocytes cultured on glass showed better colonization and survival during the first 3 days of culture. Further molecular characterization revealed evidence of accelerated epidermal differentiation in keratinocytes cultured on glass. Henceforth, functional characterization revealed that glass enhanced the temporal angiogenic and migratory capabilities of keratinocytes. Our findings pro