Fabrication of an in vitro Skin Model Aimed at Cosmetic Testing Applications

In the past, it was a common practice to test cosmetics and skin relevant chemicals directly on humans or animals, as testing protocols lacked standardised and ethical guidelines were not established. As a result, the emergence of alternative in vitro models produced today allow for safer, controlled, and a more ethical responsible pathway for drug and cosmetic testing. This thesis focuses on the fabrication of a trilayered in vitro skin model within a laboratory environment as a platform for testing cosmetics. Commercial in vitro skin models are available but are limited to simpler monolayers models or basic bilayered constructs that fail to replicate the complex architecture and function of human skin. In attempt to better recreate the functionality of human skin within a laboratory environment, various photocrosslinkable hydrogels were investigated for their ability to support appropriate cell-material interactions. However, bulk hydrogel scaffolds, commonly used by research groups to fabricate in vitro skin models, present limitations, such as the lack of nutrient diffusion through a bulk crosslinked hydrogel. Therefore, this thesis explores alternative biofabrication methods of scaffolds that support appropriate cell-material interactions. This thesis investigated the development of single layer skin models into a more representative trilayered skin construct. Key aspects of hydrogel selection and material properties were evaluated to improve keratinocyte and fibroblast proliferation, relevant differentiation, and matrix deposition. It was found that collagen I, 3 and 4 gene expression levels were higher for bilayered models, as opposed to single layered models. This highlighted the importance of including appropriate cell types and architecture to better represent human skin. By using different biofabrication techniques, microgels made from 6% HM gelMA were crosslinked into a 3D scaffold and supported some formation of collagen 4 networks, which are characteristic of subcutaneous adipose tissue. Diffusion (demonstrated by diffusion coefficient analysis) was improved within the microgel scaffold compared to the bulk hydrogel control. Overall, this research supports the development of more complex in vitro skin models, which can be used for testing cosmetic products. This was demonstrated by the application of eyeliner to the fabricated trilayered model.<p></p>