Related Experiment Video
Updated: May 12, 2026
Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium
Published on: March 9, 2018
Development of the microglial phenotype in culture
1Department of Cell Biology and Molecular Medicine, University of Szeged, Szeged, Hungary.
Abstract:
Selected morphological, molecular and functional aspects of various microglial cell populations were characterized in cell cultures established from the forebrains of E18 rat embryos. The mixed primary cortical cultures were maintained for up to 28days using routine culturing techniques when the microglial cells in the culture were not stimulated or immunologically challenged. During culturing, expansion of the microglial cell populations was observed, as evidenced by quantitative assessment of selected monocyte/macrophage/microglial cell-specific markers (human leukocyte antigen (HLA) DP, DQ, DR, CD11b/c and Iba1) via immunocyto- and histochemistry and Western blot analysis. The Iba1 immunoreactivity in Western blots steadily increased about 750-fold, and the number of Iba1-immunoreactive cells rose at least 67-fold between one day in vitro (DIV1) and DIV28. Morphometric analysis on binary (digital) silhouettes of the microglia revealed their evolving morphology during culturing. Microglial cells were mainly ameboid in the early stages of in vitro differentiation, while mixed populations of ameboid and ramified cell morphologies were characteristic of older cultures as the average transformation index (TI) increased from 1.96 (DIV1) to 15.17 (DIV28). Multiple immunofluorescence labeling of selected biomarkers revealed different microglial phenotypes during culturing. For example, while HLA DP, DQ, DR immunoreactivity was present exclusively in ameboid microglia (TI<3) between DIV1 and DIV10, CD11b/c- and Iba1-positive microglial cells were moderately (TI<13) and progressively (TI<81) more ramified, respectively, and always present throughout culturing. Regardless of the age of the cultures, proliferating microglia were Ki67-positive and characterized by low TI values (TI<3). The microglial function was assessed by an in vitro phagocytosis assay. Unstimulated microglia with low TI values were significantly more active in phagocytosing fluorescent microspheres than the ramified forms. In vitro studies on microglial population dynamics combined with phenotypic characterization can be of importance when different in vivo pathophysiological situations are modeled in vitro.
Insights
Microglial cells in culture change from ameboid to ramified, with early ameboid forms showing higher phagocytosis. This dynamic is key for modeling brain conditions in vitro.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the resident immune cells of the brain, exhibit diverse phenotypes and functions.
- Understanding microglial behavior in vitro is crucial for modeling neurological diseases.
Purpose of the Study:
- To characterize morphological, molecular, and functional changes of microglial cells in long-term primary culture.
- To investigate the relationship between microglial morphology, phenotype, and phagocytic activity over time in vitro.
Main Methods:
- Primary cortical microglial cultures from E18 rat embryos were maintained for up to 28 days.
- Immunocytochemistry, histochemistry, Western blot analysis, and morphometric analysis were used to assess microglial markers (HLA DP, DQ, DR, CD11b/c, Iba1, Ki67) and morphology (Transformation Index).
- In vitro phagocytosis assays were performed to evaluate microglial function.
Main Results:
- Microglial cell populations expanded significantly, with Iba1 immunoreactivity increasing ~750-fold and cell numbers rising >67-fold from DIV1 to DIV28.
- Microglia transitioned from ameboid to mixed ameboid/ramified morphologies, with the Transformation Index increasing from 1.96 to 15.17.
- Early ameboid microglia (TI<3) expressed HLA DP, DQ, DR and showed higher phagocytic activity compared to later, more ramified forms.
Conclusions:
- Cultured microglia undergo significant morphological and phenotypic changes, impacting their functional capabilities.
- The study highlights the importance of considering microglial age and phenotype in vitro for accurate modeling of in vivo conditions.
- Early-stage, ameboid microglia in vitro are highly phagocytic, suggesting a distinct functional role compared to their ramified counterparts.
