Methodological aspects of ex vivo-generated monocyte-derived microglia-like cells in children with autism

Current research findings suggest that dysregulation between the nervous and immune systems is a primary factor contributing to both initiation and progression of autism spectrum disorders (ASD). Microglia consists of resident brain immune cells playing a pivotal role in neuroinflammation and synaptic plasticity impairment. However, understanding these mechanisms has been limited by the lack of relevant experimental models. One promising approach involves microglia-like cells derived from peripheral blood monocytes (MDMi), which can serve as an effective model for studying human microglial function and developing personalized therapeutic strategies for neuropsychiatric diseases. The aim of our study was to optimize a protocol for generating sufficient quantities of microglia-like cells from peripheral blood monocytes of children with autism suitable for functional testing. Peripheral blood samples were collected from 18 children diagnosed with autism (mean age: 8.2 years; range: 6-12 years; 12 boys, 6 girls). Mononuclear cells were isolated using Ficoll density gradient centrifugation. After a 24-hour incubation period, adherent fractions enriched in monocytes were obtained via plate adherence. The monocytes were then cultured for either 10 or 14 days in serum-free RPMI-1640 medium supplemented with antibiotics and various cytokine cocktails to induce differentiation into MDMi. Viability was assessed using supravital staining with DAPI, while phenotyping was performed by flow cytometry targeting specific microglial markers such as P2RY12 and TMEM119. Statistical analyses were performed using PAST software version 4.03. Our findings demonstrate that successful induction of microglia-like cells requires, at least, a ten-day culture period with > 3 × 10⁵ cells per well in RPMI-1640 medium containing stable glutamine, antibiotics, and a cocktail of three cytokines: GM-CSF, 10 ng/mL; IL-34, 100 ng/mL; IL-3, 10 ng/mL. Under these conditions, approximately 6 × 10⁴ viable cells are generated, with over 90% expressing the microglial marker P2RY12. This cell population provides a valuable tool for studying ASD pathogenesis and identifying personalized treatment approaches.