Multiparametric analysis of embryonic, glial and endothelial cells cultured in microfluidic systems in standard or perturbed states

Microfluidic devices have been applied to perform the functions required in assisted reproduction, including culture and mechanical manipulation of embryos and oocytes. However, this technology is not yet established in the field, being the impact of the microfluidic culture on the health and developmental competence of in vitro-derived embryos still uncertain. In this project, a novel user-friendly microfluidic platform for the culture of murine zygotes was developed to produce viable and competent blastocysts, and to investigate potential effects of microfluidics and material toxicity on embryo development and metabolic activity. Culture medium analysis by untargeted metabolomics provided, for the first time, enhanced understanding of embryo metabolic activity and identification of metabolite changes throughout embryo development in the device, compared to traditional microdrops. Culture was also improved by using mouse uterine epithelial cells-conditioned media (CM), which significantly (p < 0.05) enhanced blastocyst development (blastocysts rates in CM were 71.8 ± 4.3%, vs. control 54.6 ± 6.6%) and had a significant impact on embryo metabolic activity and gene expression. Microfluidic techniques have also been applied to recreate in vitro models of the blood brain barrier (BBB), allowing for co-culture of several BBB cell types. However, those models often require the use of complex liquid handling systems and suboptimal flow rates. In this work, a simple microfluidic model was developed to study BBB endothelial cells and to provide optimal shear stress without the need of complex instrumentation. It was shown that endothelial cells cultured in 200 µm-wide microchannels using a flow rate of 5 µl/min were able to align and elongate in the direction of flow. The work described in this thesis demonstrates that microfluidic devices offer an attractive solution for in vitro toxicity studies and can be successfully coupled with analytical techniques for morphokinetic, genetic and metabolomic studies.