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HOW TO CULTURE KERATINOCYTES ON LAMININ SUBSTRATES
The basement membrane composition in the epidermis
Human epidermal keratinocyte (HEK) cells are the major cell type of the epidermis, the outermost layer of the skin. HEK cells maintain the barrier function of the skin and make up a major component in the wound healing process. The keratinocytes arise as basal cells at the innermost layer of the epidermis where they are positioned on a specialized basement membrane (BM) composed of a set of distinct proteins, type iv, and xviii collagens, perlecan, agrin proteoglycans, as well as laminin (LN) proteins. Following cell division, they migrate into the upper epidermal layers, ultimately terminally differentiate to form the dead surface layer of the skin (Eckert and Rorke, 1989).
Skin-specific laminin proteins maintain keratinocyte phenotypic integrity and promote cell survival
The BM underlying the neonatal and adult epidermis is highly enriched in the a5 laminin isoforms (LN511 and LN521). In a scientific article in Scientific Reports (Iriyama 2020) The authors examine how photoaging affects the function of Inter-follicular epidermal stem cells (IFE-SCs) which regulate epidermal proliferation and differentiation. The authors found that sun-exposed skin showed a decrease of MCSP-positive and β1-integrin-positive cells concomitantly with a decrease of laminin-511 at the dermal-epidermal junction (DEJ), as compared with sun-protected skin. Higher levels of laminin-511 were associated with increased colony formation efficiency, higher expression levels of MCSP as well as other stem cell markers in human skin. UVB exposure to cultured human skin impaired laminin-511 integrity at the dermal-epidermal junction and reduced MCSP-positive basal epidermal cells as well as K15-positive cells. Pouliot and colleagues demonstrated within an in vitro cell adhesion wound-healing assay that the human recombinant laminin cell culture substrates Biolaminin 511 (LN511) and Biolaminin 521 (LN521) are both potent adhesive substrates for both neonatal and adult keratinocytes and that this cell-matrix adhesion is mediated by the α3β1 and α6β4 integrins (Pouliot, 2002).
A feeder-free, xeno-free and fully defined method to culture human epidermal keratinocytes
In a recent Nature Communications article, LN511 was shown to enable robust, feeder-free, and completely xeno-free and defined long-term expansion of primary adult human dermal keratinocytes. This culture system is comparable to the 3T3-J2 feeder co-culture system in terms of colony-forming efficiency, basal marker profile, genetic stability, and the ability to form a normal stratified epidermal structure in both in vitro and in vivo models (Tjin, 2018). LN511 cultivated HEK cells also demonstrate a significantly reduced pro-inflammatory signaling pathway expressions compared to classical feeder culture-based systems. This ground-breaking article has been followed up by an article in Nature Protocols (Tjin, 2020) where the authors show that it only takes between 7 and 14 days to obtain an initial culture and once the cells reach confluency and that a secondary culture from the primary culture can be expanded up to 20-fold within 4–5 days. This new system may not only provide safer keratinocyte use in the clinics but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine.
The cell‐matrix interactions play a critical role in keratinocyte cellular behavior, wound repair and regeneration
The epidermal BM also contains laminin 332 which has been shown to supports stable anchoring of basal keratinocytes to the epidermal basement membrane and functions as a cell motility factor during wound healing. Mutations in the genes that encode the laminin 332 subunits abrogate or perturb the functions of laminin 332 which may cause disruption of the epithelial adhesion complex, resulting in a skin blistering disease known as epidermolysis bullosa. Laminin 332 promotes the fast-spreading of epithelial cells and functions as a motility factor for wound healing. Laminin 332 also plays a key role and cancer invasion.
In a nature communications article from 2018 (Ishihara, 2018), the authors show that multiple laminin isoforms promiscuously bind to growth factors (GFs)with high affinity, through their heparin-binding domains located in the α chain laminin-type G (LG) domains. The authors explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse and demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improve retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious.
LAMININ KEY ADVANTAGES
Defined and animal origin-free cell culture matrix for clinical compliance.
A biologically relevant culture environment for epithelial cells
The epithelial cells are resting on a basement membrane that contains high levels of laminin-521, laminin-511, and laminin-332.
Laminin-511 and laminin-521 are potent adhesive substrates for both neonatal and adult keratinocytes and that this adhesion is mediated by the α3β1 and α6β4 integrins.
Laminin-332 promotes a rapid spreading of epithelial cells and functions as a motility factor for wound healing and cancer invasion.
No lot-to-lot variability for standardized experiments with less variation.
Scientifically validated in high-impact journals.