Ontwikkeling van een hoge-doorvoer compatibel sferoïdeplatform ten behoeve van geneesmiddelenscreening

The early stages of drug discovery and pre-clinical studies rely on 2-dimenional (2D) in vitro cell models to screen compounds, assess cellular responses, and identify promising therapeutic targets before progressing to the clinical trials. However, 90% of drugs that successfully pass the pre-clinical phase fail when tested on human subjects26, 27, in part because these 2D cell models do not accurately recapitulate the human in vivo environment and therefore often fail to predict complex tissue-specific responses, drug metabolism, and toxicity profiles observed in actual human physiology. 3D cell models incorporate more of the in vivo physiology by mimicking the 3D architecture, cell-cell interactions and gradients of oxygen, nutrients and signalling molecules. Therefore, they provide a more physiologically relevant environment for studying drug responses. Recent advances in tissue engineering, microfabrication, and genetic engineering have facilitated the development of increasingly complex and physiologically relevant 3D cell models. However, as model complexity increases, there is a corresponding departure from the key advantages that made 2D cell culture widely adopted; ease of use, cost-efficiency, and compatibility with high-throughput screening (HTS) platforms. Among the diverse array of 3D cell culture models, spheroid culture stands out due to its relative simplicity, thereby increasing its potential for widespread adoption within the drug discovery pipeline due to its greater compliance with the advantages characteristic of 2D cell culture. In this thesis, we detail the developed and utility of the SmartSpheroPlate (SSP), a micropatterned PEG-based hydrogel platform integrated into standard glass-bottom 96-well plates that contains design features that improve its ease-of-use, cost-efficiency and HTS compatibility. The system incorporates imprinted microwells containing contact points to the glass that facilitate partial spheroid immobilization, improving tissue stability during routine. The hydrogel's high optical transparency, open-well architecture, and tuneable properties support high-resolution imaging and uniform exposure to soluble factors. The custom elastomeric stamping mechanism was developed to enable rapid, reproducible fabrication of uniform microwell geometries. To highlight the advantageous properties of the SSP, we utilized it to develop two novel methodologies in the context of insulin secretagogue screening and drug screening for Autosomal Dominant Polycystic Kidney Disease (ADPKD). For the insulin secretagogue screening, we used the SSP for generating homogeneously shaped pancreatic β-cell spheroids in using MIN6 insulinoma cells. The uniform shape and positioning of the individual spheroids enabled the simultaneous, real-time imaging of Ca2+ signals in up to 1824 independent spheroids in response to glucose and various test compounds. Using this approach, we show that increasing glucose causes concentration-dependent Ca2+ oscillations in individual spheroids, that these Ca2+ oscillations are sensitive to modulators of ATP-sensitive K+ channels, and that the frequency of Ca2+ oscillations correlate with insulin secretion. Finally, we demonstrate that the neurosteroid pregnenolone sulphate, an agonist of the cation channel TRPM3, increases the frequency of glucose-induced Ca2+ oscillations and enhances insulin release; the TRPM3 antagonist isosakuranetin inhibited these responses. For ADPKD drug screening, we utilized the SSP to develop a novel methodology by generating ADPKD spheroids using cystic cells derived from ADPKD patient biopsies. We further describe an automated pipeline for quantifying cystic volume within these spheroids using confocal microscopy. Utilizing our method, we demonstrate that cystogenesis can be modulated by pharmacological compounds and that cyst formation is, in part, independent of cellular proliferation. Notably, we show that the purine analogue azathioprine significantly attenuates cystogenesis under various culture conditions. In addition, our assay captures the genetic variability between patient-derived samples, enabling the assessment of inter-individual differences in cystogenesis and drug response. Finally, the assay protocol was partially optimized by incorporating carboxyfluorescein succinimidyl ester (CFSE) as a fluorescent marker, thereby reducing both total assay duration and imaging time. In summary, this work highlights the utility of the SSP, demonstrating its unique design features and applicability in high-throughput drug screening. The data presented aims to provide compelling evidence for the broader integration of 3D cell culture models into the drug discovery pipeline, thereby addressing critical limitations associated with traditional 2D systems.