Journal Article

797 Evaluating the functional plasticity of iPSC-derived macrophages through phagocytosis and cytokine signaling in monoculture and co-culture systems

Savic Christie, Carlson, Coby, Fiene, Rebecca, Curtis, Michelle, Twaroski Kirk, Donegan Madelyn

2025

Overview

BackgroundMacrophages modulate immune activity through cytokine and chemokine release in response to stimuli. These cytokines signal tissue and immune cells to elicit a pro- or anti-inflammatory response. iCell® Macrophages 2.0, which are derived from human induced pluripotent stem cells (iPSC), are functionally naïve and offer an effective, dynamic response to stimulation for a biologically relevant model of human macrophage function.MethodsiCell Macrophages 2.0 can be used directly out of thaw or plated and maintained for up to 14 days in culture. To measure cytokine release, cells were plated and stimulated with pro-inflammatory (LPS, IFNγ) or anti-inflammatory (IL-4, IL-13) stimuli across multiple doses and at different timepoints. Supernatants were collected and cytokines quantified by different immunoassay technologies, including Luminex, ELISA, Lumit, or HTRF. For phagocytosis, iCell Macrophages 2.0 were plated for 3 days, then incubated with pHrodo-labeled bioparticles and monitored for uptake. Antibody-dependent cellular phagocytosis (ADCP) assays were run with stimulated macrophages to measure the potency of Rituximab for CD20+ Raji target cells. iCell Macrophages 2.0 were cultured with isogenic iCell Hepatocytes 2.0, iCell Sensory Neurons, or iCell Cardiomyocytes2 to evaluate the functional effects of macrophage stimulation on complex culture systems.ResultsiCell Macrophages 2.0 express macrophage surface markers such as CD68, CD11c, and CD11b. These full function macrophages can be polarized toward a pro- or anti-inflammatory state, with cytokine release comparable to human primary macrophages for IL-6, TNFα, IL-10, and CCL18. This response can be measured within hours in both mRNA and secreted protein and can be detected at stimulant concentrations below 100pg/ml. Phagocytosis is readily measurable in both short- and long-term culture. iCell Macrophages 2.0 have a functional ADCP response to antibody-labeled target cells with a larger dynamic range than primary monocyte-derived macrophages. Proinflammatory iCell Macrophages 2.0 are able to sensitize hepatocytes to immune-mediated hepatotoxic compounds, while sensory neurons and cardiomyocytes have altered functional outputs to polarized macrophages.ConclusionsHuman iPSC-derived macrophages provide a constant, reproducible source of biologically relevant material. iCell Macrophages 2.0 are in a naïve functional state, able to respond to pro- or anti-inflammatory stimuli. Functionally, cytokine release and phagocytosis assays can be performed anytime from thaw up to 14 days in culture with consistent, reliable lot-to-lot performance. These cells are compatible with complex culture conditions across numerous tissue types. The functional profile of iCell Macrophages 2.0 demonstrates their suitability as a continuing source of human macrophages.

• Publisher: BMJ Publishing Group LTD
• Subject: Cytokines