(160a) Develoment of a Novel 3D Bilayer Skin Model: Towards a Defined Skin Equivalent for RAPID Animal Free Cosmetics Screening

Over the past three decades the development of human skin substitutes in the field of tissue engineering and regenerative medicine has significantly advanced. The manipulation of novel biomaterials has enabled the recapitulation of features of the native skin physiology including its’ biological architecture. The fabrication of skin substitutes is a very promising tool of clinical significance for wound healing, as well as of industrial significance for screening of the cosmetics and drugs. Research into the field of 3D tissue models as ‘living skin equivalents’ has been ongoing since the 1970’s with models based on Collagen I hydrogels being the first to occur¹. Since then different 3D techniques like spheroids, hydrogels and polymeric scaffolds have been used to incorporate different cell types including fibroblasts and keratinocytes ^2,3,4. However, a key limitation for most of these 3D models is that they are unable to mimic the structural differences between different skin layers, i.e., the epidermis and dermis layers. Structurally, the epidermis is a low porosity barrier for the skin with keratinocytes as its main cellular component while the dermis is a much more fibrous and porous structure containing an abundance of various ECM proteins like Collagen I and cellular components like fibroblasts and endothelial cells.

In the present study, we aim to create a hybrid in vitro model of skin, which recapitulates the structural and cellular configurations of the epidermis and dermis, i.e., keratinocytes for the epidermis layer and fibroblasts for the dermis layer. The low porosity epidermis layer was constructed with a Collagen I hydrogel while the porous dermis layer was constructed with Collagen I coated polyurethane (PU) polymeric scaffolds^5,6,7.

Experimental methods:

Porous polyurethane (PU) scaffolds were prepared via Thermal Induced Phase Separation (TIPS) method and was surface modified with Collagen I via passive surface absorption for extracellular matrix (ECM) mimicry^5,6,7. The PU scaffold was populated with fibroblast cells to mimic the dermis layer. Keratinocytes were cultured on to Collagen I containing Peptigel (Manchester BIOGEL, UK) to mimic the epidermis layer. The two sections were cultured individually for 1 week, post which they were assembled to for a 2-layer hybrid model of skin equivalent followed by long term culture of 3 weeks in a liquid- air culture method. Various in situ assays to measure and visualize cellular growth, proliferation, spatial organization and ECM mimicry was carried out at specific time points.

Results:

We have developed a long term bi-layer hybrid model of ‘skin equivalent’ focusing on mimicry of porosity and cellular component differences between epidermis and dermis layer of the skin. The developed model shows cellular growth, aggregation, proliferation as well ECM secretion for several weeks. Cellular interaction and cell motility across the two layers was also observed.

Conclusion:

Our bilayer hybrid 3D ‘skin equivalent’ captures keys structural and cellular differences between skin layers and can be considered as a platform for various applications ranging from basic biological studies to applications in the cosmetics and pharmaceutical industries, i.e., via screening of the diffusivity and cell impact of cosmetic products and drgus.

Acknowledgments:

This work was supported by the Royal Academy of Engineering via an Industrial Fellowship to E.V.. Further financial support was received from the Department of Chemical and Process Engineering of the University of Surrey, an Impact Acceleration Grant (IAA-KN9149C) from the University of Surrey, an IAA–EPSRC Grant (RN0281J) and the Royal Society. P.G has received financial support from a Commonwealth Rutherford Post-Doctoral Fellowship (2018 – 2020) and the 3D BioNet/ UKRI (2020-2021).

References:

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6. Gupta, et al. RSC Advances.2019; 9 (71): 41649-41663
7. Totti, S. et al. Sensors and Actuators B: Chemical, 296, 126652.