<b>Abstract</b>— <p>Microglia 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-glial 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 protein vimentin, 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, expression of genes encoding the mentioned pro-inflammatory proteins was suppressed, 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 Ca²⁺ concentration in microglial cells. Thus, the described effects of LPS may indicate that the combination TGFβ + MCSF + cholesterol added to the culture medium promotes the preservation and proliferation of functionally active microglial cells in neuron-glial cultures.</p>