Development of an image-based high throughput screening platform in full thickness skin

Both academic and industrial groups involved in drug discovery use a technique called high-throughput screening (HTS) in order to identify compounds that can be developed into drugs. HTS is a method in which a researcher can quickly conduct millions of "tests" to identify compounds that are active in a particular assay. The assay is typically prepared by growing a relevant type of cell in a plate with hundreds of wells. Each well is treated with a different compound and then evaluated for a desired change. Although this method has led to a significant number of pharmaceuticals that are currently available for use, there are a high percentage of 'hit" compounds that do not result in the desired outcome in more complex systems such as whole tissue or even animals.

Researchers have tried to prepare more advanced models by growing different types of cells in a specialised, sterile environment and then combining them in the appropriate sequence at the right time intervals to create what are termed "tissue equivalents". Although these models are useful for certain studies, they are not an accurate model of living, intact tissue when it comes to complex processes. Other models take advantage of tissue that is discarded following surgical procedures, such as skin. However, once the skin has been removed from the body, without swift corrective action it rapidly begins to die and is typically unable to respond to external stimuli within 24 hours. We have developed a skin model in which we quickly re-stretch and maintain the skin at its original tension. Remarkably, keeping it under optimal tension greatly increases the lifetime of cultured skin such that we can perform experiments lasting up to even one full week. Here, we plan to optimise this system to allow ground-breaking drug discovery research through the use of a cutting-edge method to visualise target proteins in the skin tissue. This imaging based drug discovery platform will be used to develop therapies for highly debilitating skin disorders.

In summary, the novel work proposed here has a high likelihood of success and will provide a system in which new dermatology drugs can be discovered and optimised for human use.

The drug discovery field has been revolutionised by the ability to perform high-throughput screening (HTS) in cell-based assays; however, the single, population-averaged readouts from monolayer cells often do not translate into tissue- or animal-based assays. We propose here to perform HTS in full thickness human skin by combining novel, state-of-the-art skin culture and imaging techniques. We have recently developed an ex vivo full thickness skin culture system capable of maintaining the physiological complexity, metabolic activity, and structural integrity of all the skin compartments (i.e. epidermis and dermis). To date, much of the development of the culture system has been performed in circular devices with a 15-18 mm diameter; however, we have shown that devices with diameters as small as 3 mm allow reproducible and consistent results up to five days when evaluated with our standard criteria (e.g., response to small molecule stimuli and wound healing). We propose to transform our explant system into a standard 384-well format to allow automated plate and liquid handling, as well as automated high content screening (HCS) confocal fluorescence imaging. To evaluate the expression of specific disease-related proteins we propose to optimise an antibody labelling and tissue clearing protocol for skin to facilitate on-plate whole-mount volumetric confocal imaging. Once the platform is in place, we propose to perform a proof-of-principle screen of ~2,000 compounds to demonstrate the capabilities of the platform.

This 18-month programme of research is aimed at benefiting the international dermatology research community and the dermatological pharmaceutical and cosmetics industries. The benefit of this research project is to provide a new technology platform for drug discovery that allows routine and reproducible ex vivo organ culture of mammalian skin (especially human skin and its closest mammalian orthologue, porcine skin) allowing for 384-well HTS in full thickness skin using a phenotypic immunofluorescent readout.

Currently, organotypic cultures of epidermal cells, grown on a range of dermal substitute matrices, are most commonly used for studies of epidermal gene expression, disease modelling, testing of drugs etc. One commercial ex vivo human skin system is available but this exhibits low activity in all criteria measured (i.e. response to known drugs; wound healing responses) and is of a very low throughput format incapable of supporting any form of drug discovery programme. Neither of these culture systems is sufficiently reproducible or of sufficient throughput to allow development of reliable high-throughput small molecule/RNAi/CRISPR screening regimes. Furthermore, the barrier properties of the epidermis, which are central to the pathobiology of eczema, psoriasis and other important skin conditions, are only very poorly recapitulated in 3-dimensional organotypic culture systems.

In contrast, the system under development here allows ex vivo maintenance of viable skin that is virtually indistinguishable from the in vivo environment and importantly, this includes skin organ culture in 384-well format that is suitable for development of high-throughput screening campaigns. This technology represents a non-incremental change in ex vivo culture of skin that will have a high impact on the understanding/modelling of skin diseases, unravelling biological mechanisms within intact skin and supporting therapy development.