Speaker
Description
"INTRODUCTION:
Extracellular matrix (ECM) protein is often used in cell culture to provide developmental cues for cells such as skin cells. This is especially important for the generation of epidermal models as without a dermal compartment the keratinocytes are reliant on the ECM coating to provide them with a foundation to adhere to and for pro-developmental cues. A major limitation of most commercially available ECM is its composition, as the majority of commercial/scientific suppliers only provide a single ECM component (such as Collagen I). This is a major limitation as this confers reduced physiological relevance in comparison to native human ECM which contains a plethora of ECM components. In addition, commercially available products are often not of human origin which severely limits their physiological relevance and therefore their overall effectiveness in cell culture. As a result, we have developed a methodology of obtaining and purifying a broad-spectrum ECM derived from primary human fibroblasts, which will confer numerous benefits not only to cell culture, but also for topical cosmetic applications and wound healing.
METHODOLOGY:
Primary human fibroblasts were expanded and seeded into a microporous Alvetex® scaffold, which provides a 3D microenvironment to allow for replication of the native conditions found in skin that serves to promote increased cells attachment and ECM deposition. In order to stimulate maximum ECM production, the skin models were cultured in a 54mm diameter Alvetex® scaffold with Dulbecco’s modified eagle medium (DMEM) with 10% non-heat treated foetal bovine serum (NHT-FBS). Once the dermal model had reached maturity after 4-weeks of culture time, the ECM was extracted from our in-house generated dermal models by a process of mechanical homogenisation of the dermis followed by re-solubilisation of the extracted ECM components using an extraction solution. This was followed by centrifugation to remove any pieces of the supporting scaffold and cellular debris/insoluble components, leaving the ECM suspended in the supernatant, which was then collected and lyophilized to produce the final purified ECM product.
RESULTS:
Analysis of extracted ECM by Western blotting and total collagen assay showed that the composition of extracted ECM contained predominantly Collagen type I as expected from native human skin. The purified ECM was then used as a coating solution for the generation of epidermal models using primary human keratinocytes in comparison to a range of currently commercially available coating matrixes It was found that this solution was capable of supporting the growth and development of a complex and stratified epidermis.
CONCLUSIONS:
Extraction of ECM from our in-house generated dermal models has been shown to be a viable method for the production of physiologically relevant human derived ECM that is capable of supporting the growth and development of a multi-layered and stratified epidermis. Overall, this has implications in cell culture model generation to provide more physiologically relevant cell culture conditions and developmental cues. Including further uses in downstream applications in the cosmetic industry whereby human collagen can be incorporated into topical cosmetics to help improve the appearance of skin aging."
20941865528
