Reconstituting central nervous system niche cues partially restores homeostatic-like features in cultured murine...

Microglia rapidly lose their homeostatic phenotype after isolation, limiting the interpretability of in vitro studies. We systematically evaluated culture conditions that reconstitute central nervous system (CNS) niche inputs to better preserve microglial homeostatic features. Neonatal mouse microglia were isolated by fluorescence-activated cell sorting, Percoll gradients, or a shaking protocol and cultured with defined cytokines (TGF-β1, IL-34, CX3CL1), extracellular matrix (collagen IV), and metabolic support (cholesterol, insulin-transferrin-selenium) under serum-free or serum-containing conditions. RT-qPCR revealed rapid downregulation of seven homeostatic transcripts (Tmem119, P2ry12, Cx3cr1, Hexb, Fcrl2, Olfml3, Tgfbr1) within 24 h across isolation methods, with further decline for a subset over 7 days. A cytokine cocktail partially restored homeostatic gene expression, with collagen IV and cholesterol further enhancing selected transcripts. Serum-free conditions favored ramified, surveillant-like microglia, whereas serum promoted amoeboid, activated-like cells, and adding defined factors preserved ramification while improving transcriptional recovery. Transcriptomic analyses demonstrated that optimized conditions shifted global expression profiles toward neonatal ex vivo microglia, upregulated most microglia-specific homeostatic genes, and downregulated inflammatory effectors. These data define a scalable, serum-free, collagen IV-based culture system with defined CNS cues that partially preserves key aspects of microglial homeostatic transcriptional and morphological features compared with conventional serum-containing culture, providing more physiologically relevant conditions for mechanistic and disease-relevant studies.