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IN VITRO HUMAN, SPHEROID, CO-CULTURE MODEL FOR PRECLINICAL ASSESSMENT OF DIRECT APPLICATION THERAPEUTICS TO PAEDIATRIC BRAIN TUMOURS Free

INTRODUCTION: Spheroid co-cultures of brain tumours and human foetal brain tissue hold promise as physiologically-relevant in vitro models with high predictive potential. Apart from modelling tumour-host interactions, the human foetal tissue serves as a control comparing cytotoxicity and neurotoxicity. Previous co-culture models have suffered from spheroid-size variability, required manual sorting and offered limited quantitation capabilities. We describe a high-throughput platform yielding reproducible co-cultures amenable to automation, quantification and screening. METHOD: The human medulloblastoma cell line UW228-3 and foetal brain tissue were marked with two cytoplasmic, fluorescent dyes (CDCFDASE, Celltrace Violet) and seeded simultaneously in 96-well ultra-low-attachment plates. Tumour and normal cells formed reproducible (d = 600 µm, CV% = 10%), single co-culture spheroids in each well. After three days, the spheroids were exposed to the cytotoxic drug etoposide (0.3-100 µM) for 48h, followed by a 48h drug-free phase. The viability of each cell type was quantified using flow cytometry and multiphoton microscopy. RESULTS: Both dyes were retained in the mixed cultures facilitating normal and tumour tissue recognition and dose-response curve computation. Despite seeding as a mix, tumour and normal cells organised in two opposing poles in the co-cultures, with a main tumour mass and some tumour cells invading the normal tissue. Etoposide levels of 10 µM were found to maximise toxicity to tumours (6.5%viability), while normal tissue viability was 40%. CONCLUSION: The reproducible platform and universal compatibility of the marking procedure make this model transferable to other tumour types and primary tissues.