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.

Summary: 30The long-term expansion of keratinocytes under serum-and feeder free conditions 31 generally results in diminished proliferation and an increased commitment to terminal 32 differentiation. Here we present a serum and xenogeneic feeder free culture system that 33 retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue 34 microenvironment is a major contributor to determining cell fate and a key component 35 of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular 36ECMs derived from human dermal fibroblasts, cultured under macromolecular 37 crowding conditions to facilitate matrix deposition and organisation, were used as the 38 basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 39 decellularisation procedure produced matrices which, by proteomics analysis, 40 resembled in composition the core matrix proteins of skin dermis. On these ECMs 41 keratinocytes proliferated rapidly, retained their small size, expressed p63, did not 42 express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming 43 efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. 44Collectively these data indicate that the dermal fibroblast-derived matrices support the 45 in vitro expansion of keratinocytes that maintained stem-like characteristics under 46 serum free conditions. 47 48 49

The production of nanofibrous materials for soft tissue repair that resemble extracellular matrices (ECMs) is challenging. Electrospinning uniquely produces scaffolds resembling the ultrastructure of natural ECMs. Herein, electrospinning was used to fabricate Bombyx mori silk fibroin (SF) and SF/halloysite nanotube (HNT) composite scaffolds. Different HNT loadings were examined, but 1 wt% HNTs enhanced scaffold hydrophilicity and water uptake capacity without loss of mechanical strength. The inclusion of 1 wt% HNTs in SF scaffolds also increased the scaffold’s thermal stability without altering the molecular structure of the SF, as revealed by thermogravimetric analyses and Fourier transform infrared spectroscopy (FTIR), respectively. SF/HNT 1 wt% composite scaffolds better supported the viability and spreading of 3T3 fibroblasts and the differentiation of C2C12 myoblasts into aligned myotubes. These scaffolds coated with decellularised ECM from 3T3 cells or primary human dermal fibroblasts (HDFs) supported the growth of primary human keratinocytes. However, SF/HNT 1 wt% composite scaffolds with HDF-derived ECM provided the best microenvironment, as on these, keratinocytes formed intact monolayers with an undifferentiated, basal cell phenotype. Our data indicate the merits of SF/HNT 1 wt% composite scaffolds for applications in soft tissue repair and the expansion of primary human keratinocytes for skin regeneration.