G-force: una funzionalizzazione biomimetica di formati high-throughput per substrati per la ricerca farmaceutica

Drug development is becoming increasingly inefficient and expensive, partly due to the poor predictivity of conventional in vitro culture models. Traditional preclinical models in which cells grow on plastic or glass lack physiological stiffness and biochemical cues. On the other hand, advanced, biomimetic organ-on-a-chip models are pricey and incompatible with high-throughput screening (HTS). Previous work in the Synthetic Physiology Lab (SPL) at the University of Pavia showed that hydrogel fabrication by automated liquid handling is a viable solution for biomimetic functionalization of high-throughput well plates. Still, surface-energy-driven meniscus formation at the well sidewalls hinders the use of this method for cell culture and microscopy. In particular, a protocol to prepare a cell culture hydrogel with the right thickness (20-200 µm) that covers the entire well of a multiwell plate (0.3-3.5 mm diameter) is missing. To enable automated fabrication of hydrogels suitable for high-throughput assays, this thesis focused on developing a centrifugation-based protocol, referred to as G-force. With G-force, we can modify the surface free energy of the hydrogel precursor phase cast in high-throughput culture well plates and generate thin flat hydrogels on the whole bottom of the wells. To validate G-force, we performed z-stack fluorescence imaging collecting the signal of fluorescent beads embedded in the hydrogels and we coded a MATLAB routine to extract the hydrogel thickness, roughness, and total 3D volume. In a batch, 40 out of 40 gels resulted in a flat interface with appropriate coverage and thickness. Further optimizing the hydrogel for optical clarity will provide a biomimetic functionalization to generate a better in vitro model and increase the drug screening efficiency in industrial pharmaceutical pipelines.