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Culturing M1 and M2 Macrophages: Media Matters

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Macrophages are a type of white blood cell derived from monocytes that are most widely known for the ability to phagocytose cell debris, pathogens, and even cancer cells. However, it is becoming clear that the role of macrophages goes beyond eliminating cellular waste. Macrophages are often used in conjunction with T cells to measure immune cell activation and cell-mediated cytotoxicity. Moreover, researchers are beginning to tap into the power of macrophages as an avenue for cancer therapeutics.

Categories of Macrophages

Macrophages are split into two broad categories (M1 and M2) based on polarization and the functional role in immune responses, in some ways mirroring Th1-Th2 polarization of T cells.

M1 macrophages are classically activated by pro-inflammatory cytokines IFN-? or LPS, and function to attack bacterial and viral pathogens. This cell type promotes inflammation and can even cause host tissue damage in a heightened immune response.

On the other hand, M2 macrophages are activated by interleukins, such as IL-4, and by TGF-?. These immune cells dampen the immune response and promote tissue regeneration and wound healing by rebuilding the extracellular matrix. However, based on the activation markers expressed and functionality, defining categories of macrophages is more complex than initially proposed. More information on this diverse group of immune cells can be found here.

Culturing Macrophages

As macrophages become more of a research interest, it is important to examine culture conditions. Often, laboratories will utilize incorrect media due to availability or under the suggestion of a colleague. Given that the culture medium can greatly affect the proliferation of macrophages as well as their metabolic function, finding the appropriate medium and supplement combination for your environment is a necessary step for reliable results. For instance, our research shows that DME/F12 is the best culture medium for human macrophages and IMDM is the best choice for mouse macrophages.

A less explored, but just as critical, concern is that different media conditions can result in different proportions of M1 to M2 macrophages. DME/F12, IMDM, and RPMI 1640 are among the media commonly used when culturing macrophages from monocytes, with the addition of various supplements. Supplementation, like culture media itself, produces different outcomes in macrophage growth and function, as well as the proportion of M1 to M2 macrophages.

How Media Selection Affects Your Research

Considering proper media and supplementation for your macrophage culture is of the utmost seriousness, not only to ensure the correct macrophage subtype is being supported, but also to set the foundation for downstream experimentation.

Just as in tissue, macrophages react and adjust to their environments in vitro. M1 and M2 macrophages do not exist exclusively of each other; rather the environmental milieu dictates the resulting population, as is similarly observed with Th1-Th2 signatures. Communication between T cells and macrophages in culture via cytokines and/or chemokines can result in phenotypic skewing of either cell type, depending on the combination of soluble factors present. If care is not taken to ensure the desired milieu is established prior to co-culture, these conditions could lead to inaccurate interpretation of T cell or macrophage response to stimuli during experimentation.

When examining the impact of M1 versus M2 macrophages in tumorigenesis and tumor maintenance, the heterogeneity of the cell population will lead to striking differences. It is well-documented that while M1 macrophages promote an anti-tumor environment, M2 macrophages produce a variety of factors that establish a pro-tumor environment; tumor-associated macrophages (TAM) are often identified as exhibiting M2-like phenotypes.

Things to Consider When Culturing Macrophages

To avoid pitfalls, it is important to know which types of culture media conditions skew the polarization of monocytes toward M1 or M2 macrophages. Below is a table that provides general information on the factors that can influence M1 or M2 skewing (more information on these factors can be found here). Flow cytometry or real-time PCR are excellent tools to identify phenotypic and genetic markers of M1 and M2 macrophages, listed in the table below.

While determining the best experimental set-up for your needs, it is important to keep in mind that similar to T cells, macrophages do not terminally differentiate into M1 or M2; they are influenced by the environmental stimuli and exhibit plasticity.

Macrophage TypeFactors that Promote PolarizationMarkers
M1IFN-γ, GM-CSF, TNFα, LPS, poly I:CMHC II, Fcγ receptors,
M2M-CSF, IL-4, IL-3, LPS, VEGFCD163, CD206, RAGE, etc.

Culture medium can further influence the polarization to M1 or M2 phenotype.  These two subtypes produce different milieus with direct consequences for your research.

It is crucial to carefully consider the media you choose and implement assays to identify the polarization of macrophages as a part of your workflow.

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