What question did this study set out to answer?

The aim is to evaluate how a combination of TGFβ, MCSF, and cholesterol affects microglial cells in rat hippocampal cultures.

February 20, 2026

Effect of the Combination of TGFβ + MCSF + Cholesterol on the Percentage and Functional Activity of Microglia in Rat Hippocampal Cell Cultures

Key Points

The aim is to evaluate how a combination of TGFβ, MCSF, and cholesterol affects microglial cells in rat hippocampal cultures.
Used neuron-glia cultures from newborn rats.
Applied TGFβ, MCSF, and cholesterol to the cultures.
Conducted RT-PCR analysis for gene expression of inflammation-associated proteins.
Examined effects of LPS and fMLP on microglial activity and cell death.
The combination increased microglial cell numbers by over twofold.
LPS exposure raised the gene expression of inflammatory markers like IL-1β and TNFα.
Hypoxia led to reduced gene expression of pro-inflammatory proteins but insignificant cell death.
LPS and fMLP exposure enhanced superoxide production and increased intracellular calcium concentrations.

Abstract

Microglial cells in the brain are considered resident macrophages possessing a number of functional and physiological characteristics typical of these immune cells. Microglia are involved in neuroinflammatory processes of various etiologies, during which they undergo phenotypic changes. In neuron–glia cultures, microglial cells typically have low proliferative capacity due to the absence of necessary growth factors. In this study, we evaluated the effect of a combination of compounds critical for microglial proliferation, such as transforming growth factor beta (TGFβ), macrophage colony-stimulating factor (MCSF), and cholesterol, on the number and functional activity of microglial cells in hippocampal cultures from newborn rats. We found that the combination TGFβ + MCSF + cholesterol increased the number of microglial cells in cultures by more than twofold. RT-PCR analysis showed that exposure to the pro-inflammatory agent lipopolysaccharide (LPS) in cultures grown using this combination of factors led to increased expression of genes encoding inflammation-associated proteins such as IL-1β, TNFα, STAT3, as well as the gene encoding vimentin protein, which acts as a situational marker of reactive microglia. Additionally, incubation with LPS led to increased cell death in the cultures. In the case of hypoxic episode exposure, suppression of gene expression encoding the mentioned pro-inflammatory proteins was observed, while the increase in cell death was insignificant. LPS, as well as chemotactic formylated peptide (fMLP, an immune cell activator), caused enhanced production of superoxide anion and increased intracellular Ca concentration in microglial cells. Thus, the described effects of LPS may indicate that the combination of TGFβ + MCSF + cholesterol added to the culture medium promotes the preservation and proliferation of functionally active microglial cells in neuron–glia cultures.

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