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Enhanced immune response of human iPSC-macrophages via animal-free manufacturing processes

ABSTRACT: Studying human macrophages in vitro is crucial for understanding their immune functions. Recent interest in macrophages derived from induced pluripotent stem cells (iPSC-Mac) has led to various differentiation protocols, often using undefined animal serum like fetal bovine serum (FBS). This study compared FBS-based media to the defined medium X-VIVO™ 15 containing human serum albumin (hSA). Both produced functional macrophages, but those in hSA displayed a neutral activation state, enhanced cholesterol metabolism, and more significant changes in surface phenotype after stimulation. They also had a stronger response to co-stimulatory signals, making them better for testing immunomodulatory drugs. In contrast, FBS-grown macrophages were more adherent and pre-activated, showing reduced sensitivity upon stimulation, which may pose challenges for drug screening and immunomodulatory evaluations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE311159 | GEO | 2026/04/22

REPOSITORIES: GEO

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Project description:Macrophages are central to innate immunity and defining the mechanisms by which these cells mediate inflammation is critical to understanding in situ homeostatic and pathophysiologic roles. Human monocyte derived macrophages (hMDM) are widely used to examine these processes. Stimuli that influence macrophage function could influence these studies, including differences in culture environment that have been suggested significantly impact macrophage function. We examined baseline hMDM activity and the response to the TLR4 agonist, lipopolysaccharide (LPS) in different culture media in the presence and absence of serum. We focused on the most common macrophage medias, DMEM and RPMI 1640 Medium +/- fetal bovine serum (FBS), as well as Macrophage Serum Free Media (M-SFM), a media specifically formulated for serum free culture. Cells cultured in these five distinct media were treated with vehicle (H20) or LPS and evaluated for changes in morphology, transcriptomic profile, and alterations in inflammatory response. Culture in M-SFM resulted in the greatest morphological differences (area, length-to-width, perimeter-to-area ratio) and while there were few transcriptomic differences between DMEM and RPMI with FBS, both the absence of FBS supplementation and culture in M-SFM significantly altered the baseline transcriptome. Culture in M-SFM also significantly reduced macrophage sensitivity to LPS as measured by NF-kB activation. All hMDM conditions showed a dose dependent phagocytic response and elicited cytokine and chemokine secretion in response to LPS stimulation, but the magnitude of these effects varied between conditions, with different trends depending on the cytokine. These findings suggest that culture media and the presence of serum alters baseline activation and the response to stimulus in macrophages, potentially obscuring or driving artifactual results. These studies confirm and expand on previous findings showing that in vitro microenvironment is not a benign component of experimental design and demonstrate the need to optimize experimental conditions for such plastic cells.

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Enhanced immune response of human iPSC-macrophages via animal-free manufacturing processes

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