Humanising drug discovery with integration of automation workflows

A current lack of physiologically relevant in vitro cell disease models has led to high attrition rates of novel therapies progressing through clinical trials. In recent years, the use of induced pluripotent stem cells (iPSCs) derived from patients has improved effective drug screening for human genetic diseases. However, there are challenges in using iPSCs derived cell types for screening programmes, such as scalability, purity of cell populations that robustly maintain disease phenotype, and variability in data across labs. We have developed a High Content Imaging (HCI) phagocytosis assay using automation systems to culture iPSC derived microglia (iMGL), increase assay throughput, and demonstrate consistent functionality which is necessary for screening and identifying compounds which might be good chemistry start points for projects focussed on neuroinflammation. Human iMGL were dispensed into 384-well imaging plates using the Multidrop™ Combi Reagent Dispenser and matured over 7 days with frequent media changes using the Integra VIAFLO. Compounds were prepared using Echo Acoustic Liquid Handlers and dispensed onto cells using the Biomek NXᴾ Automated Workstation. Cells were then treated with pHrodo Red-labelled Amyloid-β 1-42 (Aβ) to stimulate phagocytosis, after which time they were fixed and stained with Hoechst to identify nuclei, and specific antibodies to a range of microglial markers. Cells were imaged using the ImageXpress Confocal HCI System and automated plate handling. Phagocytosis was quantified using a Cell Scoring algorithm in MetaXpress6 to identify cells positive for pHrodo-Aβ near the nucleus. Mosaic was used for compound tracking and Genedata for data analysis. To characterise the consistency of iMGL populations, we evaluated the expression of the microglial markers transmembrane protein 119 (TMEM119), purinergic P2Y12 receptor and triggering receptor expressed on myeloid cells 2 (TREM2). A comparison between manual and automated assays confirmed that there was no significant difference in the number or morphology of cells, the expression of microglia markers using either approach. Similarly, there was no significant difference in the percentage of phagocytosis positive iMGL, which was approximately 50 % for both assay formats. In conclusion we have established an automation process to support culture of iPSC derived cells and a HCI phenotypic assay to both screen for novel compounds that augment phagocytosis and explore iMGL cell biology. These processes can be adapted to a range of different assay formats suitable for neurodegenerative disease research and will allow us to accelerate human neuroscience drug discovery.