Developing a 3D bio-printed human skin model

Unravelling the pathophysiological mechanisms of skin disease relies on representative skin models. However, current laboratory skin models have acknowledged limitations which impede translation to the clinic. The need for a stratified 3D cellular co-culture with control over spatial organization to represent the complexities of human skin more realistically is therefore highly desirable. 3D bio printing has recently generated physiologically relevant human skin models (Baltazar et al. 2020). However, current bio printing technologies are typically expensive, difficult to operate, and have low customisation ability, thus hindering widespread accessibility (Ioannidis et al. 2020). Custom-built, low-cost 3D bio-printing platforms have been recently reported for the production of 3D cell culture and tissue models (Cubo et al. 2016a; Reid et al. 2016; Kahl et al. 2019; Ioannidis et al. 2020). It is therefore hypothesised that recreating the structure of human skin through developing a cost-effective flexible 3D bio-printing technology is feasible. The aim of this study is to develop a 3D-bio-printed human skin model using a low-cost flexible cell-printing platform. Preliminary 2D cell culture studies were conducted using an immortalized keratinocyte cell line to establish the optimum culture conditions. Cells were maintained in a proliferative or differentiated state by varying the calcium concentration to mimic the physiological epidermal calcium gradient (Wilson et al. 2007; Bikle et al. 2012). Morphology and specific biochemical markers of differentiation were studied in each condition. A bespoke LEGO® 3D bio-printer, capable of encapsulating high cell densities and creating 2D and 3D arrangements of cells, was built in parallel to the cell culture experiments. Cells maintained in low calcium exhibited proliferative characteristics whereas cells in higher concentrations of calcium were induced to become more differentiated, recapitulating the effect of the calcium gradient in the epidermis. The programmed custom-built LEGO® 3D bio-printer was optimized to generate high-resolution 2D and 3D complex patterns of bio-ink. Using the custom-built 3D bio-printer, the cells were successfully encapsulated in bio-material droplets and printed. Microscopy images and a cell viability assay indicated homogenous cell dispersion and high cell viability (87.5%) within the bio-printed material. Keratinocytes were successfully 3D bio-printed in an 18-layered squared lattice and imaged showing high cell viability. These initial results provide a platform for manufacture of single and mixed cell ii culture populations with a defined 3D organization, akin to the human skin. The adaptability and flexibility of the custom-built LEGO® 3D bio-printer has the potential to enhance the complexity of the skin tissue model. Therefore, a first prototype of the LEGO® 3D bio-printing platform has been developed demonstrating a printing resolution at the sub-millimeter scale, providing a cost-effective novel 3D bio-printing technology for the production of human skin models