TetraCultures of Neurons, Astrocytes, Oligodendrocytes, and Microglia: A Murine In Vitro Model to Dissect Region-Specific CNS Immune Dynamics

Neuroinflammation and glial scar formation in the central nervous system (CNS) emerge from coordinated signaling between neurons, astrocytes, oligodendrocytes and microglia. Conventional embryonic neuron-astrocyte-oligodendrocyte (NAO) TriCultures lack microglia and cannot model innate immune responses. Here, we introduce postnatal neuron-astrocyte-oligodendrocyte-microglia (NAOM) TetraCultures, in which resident microglia are retained from the original brain tissue and co-develop with other CNS cell populations from the onset of culture. Hippocampal and cortical cultures were maintained for 14 days in vitro and challenged with LPS, poly I or scratch injury. Only NAOM TetraCultures mounted robust, region- and stimulus-specific inflammatory responses. LPS induced marked TNF-αand IL-1βsecretion and increased IBA1 in hippocampal microglia, whereas poly I elicited IFN-βrelease without increasing IBA1. CD68 regulation differed between hippocampus and cortex, and microglial morphology showed pronounced amoeboid transitions after LPS. Astrocytes in NAOM TetraCultures expanded their territorial coverage after LPS, an effect absent in NAO TriCultures. Following scratch injury, IL-1βincreased, microglia infiltrated lesion zones and astrocytes formed lesion borders. Thus, NAOM TetraCultures recapitulate key features of CNS immunity, glial scar formation and region-specific glial responses while providing a physiologically relevant microglia-containing model.

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Authors

Lea Gabele-Kutsche; Yaning Zhao; Tabea Spengler; Kay Michael Gottlieb; Christian Deppe; Martin Korte; Kristin Michaelsen-Preusse

Published

Oct 12, 2026

Paper type

preprint

Source

Arxiv