Bioprinting of skin equivalents for immunotoxicity testing

Abstract

The aim of this project was to develop bioprinted 3D full-thickness human skin equivalents (HSEs) which could be exploited for safety testing of biologics in-vitro. In this study, handmade 3D HSEs in a 24-well format were first optimised, and the structure of the 24-well HSEs were shown to be comparable to physiological healthy human skin. To improve the feasibility of bioprinting 3D HSEs the overall size of the equivalents was reduced by scaling the HSEs to a 96-well plate format using prototype 96-well Alvetex® inserts. Histological analysis of the optimised 96-well skin equivalents showed these to be similar to both the 24-well HSEs and physiological human skin. To demonstrate that skin cells could be bioprinted in the high densities required to create 3D HSEs, cells were printed using a Jetlab 4 Tabletop printer and solenoid microvalves. Fibroblasts were printed at concentrations of 1x106 cells/mL, 10x106 cells/mL, 20x106 cells/mL and 40x106 cells/mL. Cell count and viability immediately post printing demonstrated that microvalves could be used to reliably dispense a high density of cells over 1 hour with minimal cell death. 96-well HSEs were then bioprinted and compared to handmade 24-well and 96-well HSEs in addition to human skin using histological techniques. Fully autologous 96-well HSEs were then bioprinted and co-cultured in-vitro with autologous peripheral blood mononuclear cells (PBMCs) in the presence and absence of biologics known to cause (OKT3) or not to cause (Natalizumab) immunotoxicity. Investigation of supernatants revealed significant proinflammatory cytokine responses (interferon gamma (IFN-γ)) in co-cultures treated with OKT3 when compared to negative controls. This research developed a 96-well format bioprinted HSE and demonstrated the feasibility of using bioprinted 3D tissue equivalents for in-vitro immunotoxicity testing of biologics. In conclusion, microvalve based bioprinting technology can be used to fabricate HSEs suitable for application of in-vitro screening of biologics.