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Microglial Cell Lines: The Ultimate Guide to Selection

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Microglia comprise the primary tissue macrophage population of the brain. As such, they dynamically survey their environment, clearing debris, remodeling neuronal circuits, and performing other sundry housekeeping duties. Although recent advances in transcriptomics have revealed key molecular differences between in vivo microglia and microglial cell lines, [1] the latter remain useful due to their amenability to high-throughput assays and genetic manipulation. But which microglial cell line should you choose for your experiments?

This article outlines the most popular and readily available microglial cell lines and offers advice on which one might be right for your purposes.

Pros and Cons of Microglial Cell Lines

Like any tissue macrophage, microglia are “educated” by the tissue in which they reside — in this case, the brain. [2] So microglia perform brain-specific functions because brain-specific factors tell them to. For example, neurons release chemokines to inform microglia about disturbances to their health. [3] When microglia are removed from the brain and put into a plastic dish, they lose these brain-specific signals and, therefore a part of their identity. [4]

Because conventional cell culture conditions can hardly replicate the complex milieu of the brain, many have argued that microglial cell lines poorly represent their in vivo counterparts. [1] This is largely true. However, in vivo microglia are notoriously difficult to manipulate genetically, and high-throughput assays are nearly impossible to perform in vivo. In addition, human microglia are ethically and technically challenging to obtain. Microglial cell lines can thus be used to complement — not replace — in vivo microglia.

The BV-2 microglial cell line provides one concrete example of the utility of microglial cell lines. It was used recently in CRISPR/Cas9 screening assays to identify genetic regulators of impaired phagocytosis and lipid droplet formation. [5, 6] These screens provided key insights into microglial biology that would have been difficult if not impossible to ascertain had a cell line not been used, since CRISPR/Cas9 screens require millions and millions of cells.

How to Choose a Microglial Cell Line

Most microglial cell lines were made before we knew much about what makes microglia unique relative to other macrophages. Arguably, the bar for microglial cell line generation — i.e., to what extent does a new microglial line recapitulate in vivo microglia? — is higher now than it was in the 1990s because we know so much more about microglia. This makes more recently generated microglial cell lines more attractive if your goal is to recapitulate microglial biology.

As when choosing any cell line, there are several factors you should consider when choosing a microglial cell line: authenticity, species, method of immortalization, functional characteristics, biosafety concerns, and medium requirements, as discussed below.

Authenticity and Species

Many microglial cell lines were generated over twenty years ago and have passed through multiple labs. Without authenticating a cell line’s identity, you cannot be certain of its origin, even when obtained from the lab that generated the line. Authenticity and species information are critical for gene perturbation experiments, since genomic sequences vary substantially across species. In addition, human microglia may be more relevant for studying human-specific diseases such as human immunodeficiency virus-1 (HIV-1) infection. [7]

Method of Immortalization

Microglial cell lines have been generated by two immortalization methods: retroviral transduction of viral oncogenes or spontaneous immortalization. Although neither truly replicates the untransformed characteristics of in vivo microglia, retroviral immortalization can cause very rapid growth rates (doubling times < 24 hours) and aberrant genomic characteristics (e.g., abnormal chromosome numbers). The growth rate of virally transformed microglia can be slowed by subsequent transduction with the human telomerase (hTERT) gene. [7] While fast-growing microglial cell lines may represent a significant departure from in vivo microglia, they come in handy for genome-scale screening applications that require millions of cells.

Functional Characteristics

Functional characteristics such as cell surface markers, responsiveness to inflammatory stimuli, and phagocytic capacity are important indicators of cellular fidelity — i.e., does a particular cell line recapitulate in vivo microglia? — and determinants of experimental success. For example, BV-2 cells have been shown to be more phagocytic than HMC3 and CHME5 cells, [8] so they may be more suitable for phagocytosis-related applications. These characteristics are discussed in more detail later on in this article.

Biosafety Concerns

Many microglial cell lines are immortalized by retroviral transduction of viral oncogenes (e.g., v-myc). Even though these cells do not propagate these viruses, they require special handling practices — e.g., those defined by the U.S. Public Health Service and the American Type Culture Collection (ATCC) — because they contain viral sequences that could be harmful to humans. Although spontaneously immortalized microglial cell lines can be handled under less stringent conditions (i.e., BSL-1), ATCC recommends using all cell lines under BSL-2 conditions because no cell line can be tested for all viruses.

Medium Requirements

Colony-stimulating factor 1 (CSF-1) is a major growth factor for many macrophage populations in vivo. As such, some spontaneously immortalized microglial cell lines require CSF-1 supplementation to maintain their growth and survival in culture. CSF-1 is not necessary for retrovirally-transduced microglial cell lines, as the viral oncogenes present in these lines are sufficient for their survival and growth.

Commonly used and/or readily available microglial cell lines are summarized in Table 1. Use this handy guide to help you decide which microglial cell line to use for your application.

Table 1: Characteristics of commonly used microglial cell lines

Cell LineSpecies Immortalization Method Growth Medium Biosafety Level (BSL) Where to obtain
BV-2 Mouse Retroviral / v-raf & v-myc DMEM + 10% FBS BSL-2 [9]
N-9 Mouse/unclear Retroviral / v-myc DMEM + 10% FBS BSL-2 [10]
CHME-5 Rat/unclear SV40 large T antigen DMEM + 10% FBS BSL-2 [11]
HMC3 Human SV40 large T antigen EMEM + 10% FBS BSL-2ATCC CRL-3304
HMC20 Human SV40 large T antigen / hTERT DMEM + 5% FBS BSL-2 [8]
EOC 2 Mouse Spontaneous DMEM + 10% FBS + 20% LADMAC conditioned media BSL-1 ATCC CRL-2467
EOC 13.31 Mouse Spontaneous DMEM + 10% FBS + 20% LADMAC conditioned media BSL-1 ATCC CRL-2468
EOC 20 Mouse Spontaneous DMEM + 10% FBS + 20% LADMAC conditioned media BSL-1 ATCC CRL-2469
SIM-A9 Mouse Spontaneous DMEM/F12 + 5% horse serum + 10% FBS BSL-1 ATCC CRL-3265
IMG microglia Mouse Retroviral / v-raf & v-myc DMEM + 10% FBS BSL-2 Sigma-Aldrich

We take a closer look at these cell lines below.

Murine Microglial Cell Lines

The BV-2 cell line, developed by Blasi and colleagues [9], is one of the most popular microglial cell lines ever created. It is also one of the oldest; it was established in 1990, through retroviral transduction with the v-myc and v-raf oncogenes. Due to its popularity, this line has been compared extensively to primary newborn as well as adult microglia. Independent studies have shown that when challenged with various inflammatory agents, BV-2 cells respond similarly to, albeit less aggressively than, primary microglia. [12]

The IMG microglial cell line was derived more recently by immortalizing adult mouse microglia in the same manner as BV-2s. [13] IMG cells respond to inflammatory agents more potently than BV-2 cells do, so they may better recapitulate the sensitivity of primary microglial cells. IMG cells also display the same phagocytic capabilities as BV-2 cells.

The N-9 and CHME-5 microglial cell lines are also somewhat popular choices. However, their authenticity is questionable, likely because of poor documentation. The N-9 cell line is commonly documented as originating from rat microglia when, in fact, the original paper clearly describes the immortalization of mouse microglia. [10] Even more worrisome, the CHME-5 cell line — originally billed as a human microglial cell line [11] — was recently found to be of rat origin by researchers at Case Western and the National Institutes of Health. [7, 14] Therefore, stocks of these cell lines obtained from other labs should definitely be authenticated prior to use to verify their identity.

The microglial cell lines discussed above were derived by genetic means. If you’re looking for a spontaneously immortalized alternative, look no further than the SIM-A9 and EOC lines. SIM-A9 cells express microglial markers such as Iba1 and CD68, respond normally to pro-inflammatory and anti-inflammatory agents, and phagocytose amyloid-? and bacterial particles. [15] The EOC microglial cell lines display similar features but require colony-stimulating factor-1 (CSF-1) supplementation during culture. [16]

Human Microglial Cell Lines

Human microglia are hard to come by. Not only do they need to be obtained from fetal sources or surgically resected/postmortem brain tissue, but they must also be isolated as quickly as possible to avoid activating them prior to use. Some researchers have resorted to immortalizing human microglia to generate a renewable source for their experiments.

Originally denoted CHME3, the HMC3 cell line was recently authenticated by ATCC after some confusion about its origins. [17] The CHME3 and CHME5 lines originated from the same laboratory, [11] but the latter was contaminated with rat cells as discussed above, probably early during its distribution. Thankfully, the HMC3 line was not. HMC3 cells possess many features of primary microglia, including microglial surface markers, phagocytic potential, and inflammatory responses to pro-inflammatory stimuli.

The HMC20 cell line was obtained from cryopreserved human microglia immortalized with the SV40 large T antigen (SV40) and the human telomerase (hTERT) gene. [7] The expression of hTERT dramatically reduces the proliferative rate of SV40-immortalized human microglia and slows the progression of cellular senescence. HMC20 cells respond normally to inflammatory stimuli, phagocytose apoptotic neurons, and express microglia-specific cell surface receptors such as P2RY12. And their basal transcriptome overlaps significantly with those of ex vivo human and mouse microglia. These features make the HMC20 line ideal for low-throughput applications that require human rather than murine microglia.

For more detailed comparisons between these cell lines, check out the reviews by Stansley et al. [12]; Timmerman, et al. [18]; and Rodhe [19].

Learn more about how to use cell lines from our article discussing the concept of biological reproducibility for cell line experiments.


1. Butovsky O, et al. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat Neurosci. 2014;17(1):131-43.

2. Gosselin D, et al. Environment Drives Selection and Function of Enhancers Controlling Tissue-Specific Macrophage Identities. Cell. 2014;159(6):1327-40.

3. Biber K, et al. Neuron-microglia signaling: chemokines as versatile messengers. J Neuroimmunol. 2008;198(1-2):69-74.

4. Gosselin D, et al. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017;356(6344).

5. Pluvinage JV, et al. CD22 blockade restores homeostatic microglial phagocytosis in ageing brains. Nature. 2019;568(7751):187-92.

6. Marschallinger J, et al. Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nat Neurosci. 2020;23(2):194-208.

7. Garcia-Mesa Y, et al. Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system. J Neurovirol. 2017;23(1):47-66.

8. Overland A, et al. Quantitative, live-cell kinetic analysis of microglial function and morphology. In: Society for Neuroscience 2018 Meeting, Nov 3-8; San Diego, CA.

9. Blasi E, et al. Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol. 1990;27(2-3):229-37.

10. Righi M, et al. Monokine production by microglial cell clones. Eur J Immunol. 1989;19(8):1443-8.

11. Janabi N, et al. Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci Lett. 1995;195(2):105-8.

12. Stansley B, et al. A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s disease. J Neuroinflammation. 2012;9:115.

13. McCarthy RC, et al. Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta. J Neuroinflammation. 2016;13:21.

14. Campbell LA, et al. Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus. Mol Ther. 2019;27(1):151-63.

15. Nagamoto-Combs K, et al. A novel cell line from spontaneously immortalized murine microglia. J Neurosci Methods. 2014;233:187-98.

16. Walker WS, et al. Mouse microglial cell lines differing in constitutive and interferon-gamma-inducible antigen-presenting activities for naive and memory CD4+ and CD8+ T cells. J Neuroimmunol. 1995;63(2):163-74.

17. Dello Russo C, et al. The human microglial HMC3 cell line: where do we stand? A systematic literature review. J Neuroinflammation. 2018;15(1):259.

18. Timmerman R, et al. An Overview of in vitro Methods to Study Microglia. Front Cell Neurosci. 2018;12:242.

19. Rodhe J. Cell culturing of human and murine microglia cell lines. Methods Mol Biol. 2013;1041:11-6.

image of microglia

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