METHODOLOGICAL ASPECTS OF MONOCYTE-DERIVED MICROGLIA-LIKE CELLS GENERATION AT CHILDREN WITH AUTISM

Abstract

Current research acknowledges that dysregulation between the nervous and immune systems is a primary factor contributing to both initiation and progression of autism spectrum disorders (ASD). Microglia is a resident brain immune cells, play 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 this 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 through plate adherence. These 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 conducted via 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 ten-day culture periods involving no fewer than 3×10⁵ cells per well in RPMI-1640 medium containing stable glutamine, antibiotics, and a cocktail consisting of three cytokines: GM-CSF, 10 ng/mL, IL-34, 100 ng/mL, and 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 investigating ASD pathogenesis and identifying personalized treatment approaches.