The application of Design of Experiment to optimise a spheroid model of kidney fibrosis

Fibrosis, the accumulation of scar tissue in response to a variety of physiological and environmental insults, can lead to progressive organ failure. The quantification of fibrosis biomarkers in in vitro models is a powerful tool for assessing novel
anti-fibrotics, but these models have historically been poor predictors of in vivo efficacy due to complicated disease provenance and pathophysiology. There is growing recognition that 3-dimensional (3D) cellular models may offer superior
predictivity by better recapitulating the complexity of diseased tissue. In this poster, we present the development of a 3D in vitro rat kidney spheroid assay to improve translation to in vivo models.
As more traditional One Factor at a Time (OFAT) optimisation of assay parameters is unrealistic for protracted fibrosis assays, we utilised a Design of Experiment (DoE) approach to evaluate multiple non-linear parameters simultaneously in a
Design Expert statistical model. A D-optimal Response Surface model was developed to determine the parameters essential for evolution of a fibrotic phenotype by NRK-49F spheroids. DoE enabled a rapid and comprehensive evaluation of the
assay design space. Testing 200 conditions across 5 numeric and 3 categorical parameters, we optimised our model for screening small molecules and validated against known TGF-b1 signalling inhibitors. Assay quality and response window
were improved over the corresponding 2D assay.