Commentary: Neuroinflammatory In Vitro Cell Culture Models and the Potential Applications for Neurological Disorders.

We read with great interest the article by Peng et al. (2021), “Neuroinflammatory In Vitro Cell Culture Models and the Potential Applications for Neurological Disorders,” which appeared in Frontiers in Pharmacology on 23 April 2021. This review was helpful for further understanding the functions and limitations of different cell lines in neuroscience research and inspired our team in selecting in vitro cell culture in the neuroinflammatory study.

However, incorrect organismic classification of BV-2 cells was discovered in the body of the article. In the section “Microglia,” the authors mentioned that “The two commonly used cell lines produced by the replacement are BV-2 and N9 cells from rats and mice to quickly produce large numbers of cells, respectively.” Actually, BV-2 and N9 cells were both retroviral immortalized microglia from mice beyond any doubt. Similar errors were found in a review written by Branden et al. (Stansley et al., 2012) in the Journal of Neuroinflammation. In the article, at multiple places, they wrongly claimed that BV-2 cells were used for rat models. Until now, none of the authors have posted explanations and corrected revisions in the journal.

The role of microglia has been a research hotspot in the field of neuroinflammation. With a time-consuming and costly culture process, and low quantities of purified cells from cultivating primary microglia, as an alternative, investigators have created several immortalized cell lines to study the function of microglia in vitro, including mice derived BV-2 (Blasi et al., 1990) and N9 cells (Righi et al., 1989), rat derived HAPI cells (Cheepsunthorn et al., 2001), and human derived HMO6 cells (Nagai et al., 2001).

Remarkably, apart from the genomic alterations that have presented them immortal, differences in variance of morphologies, adhesion properties, and proliferation rates have also been observed compared to primary microglial cells. Despite these differences, some researchers continue to use them in experimental culture models under the assumption that they represent primary microglial function to some extent. Among these immortalized cell lines, BV-2 cells derived from newborn brain of inbred C57BL/6 mice by infection with the J2 retrovirus carrying the v-raf/v-myc oncogene have been most extensively used as an in vitro culture scheme, including experiments in studying LPS-induced inflammation and classical neurotoxicity, detecting cytokine secretion and cell-surface receptors (e.g., purinergic receptors), examining certain signal molecules and signal pathways (e.g., ERK and MAPK signaling), and even using in electrophysiological studies. Nevertheless, BV-2 cells do not fully express primary microglia characteristics, and some microglia-specific genes were rarely expressed in microglia cell lines (Butovsky et al., 2014). Several comparative studies have revealed differences in molecular signature between these immortalized cell lines and primary microglia (for review, see Henn et al. (2009) and Das et al. (2016)).

Besides BV-2 cells, also other microglia cell lines were available, although they were less frequently used. Most notable were the N9 cells derived from embryonic brain of outbred ICR/CD1 mice by culture with the 3RV retrovirus carrying an activated v-myc/v-mil oncogene of the avian retrovirus MH2, which were habitually employed as parallel control together with BV-2 cells or used in combination with primary microglia to validate the inflammatory response intracellular signaling pathways synergistically. N9 cells shared many phenotypical characteristics with primary microglia, but not to the same extent, including the release of inflammatory cytokines by LPS and the expression of microglial cell surface markers (e.g., positive expression of FcR, Mac-1, and F4/80, negative of GFAP, A2B5, and Gal-C) and receptors (e.g., purinergic receptors) (Stansley et al., 2012).

Although N9 cells have also been shown to be similar to BV-2 cells, a few reports of differences between BV-2 and N9 cells have been published. Collectively, several studies have demonstrated that BV-2 cells have intense phagocytic activity, yet they lack robust secretory activity. In contrast, the N9 cells have been shown to possess the solid secretory activity and produce reactive nitrogen intermediates and cytokines such as TNF-α, IL-6, MCP-1, and IL-8 (Righi et al., 1989; Meda et al., 1995; Zhang et al., 2003; Nikodemova and Watters, 2011). In addition, BV-2 cells were more sensitive to NGF and BDNF than N9 cells (Zhang et al., 2003). Since both neurotrophic factors boost the proliferation and survival of microglia, this result may suggest that BV-2 and N9 cells have diverse potential performances in regulating neuronal cells during CNS repair. Differential immune responses of BV-2 and N9 cells may be due to different immortalization techniques and mice of different genetic backgrounds used to derive these cell lines. There have also been descriptions of other microglia cell lines, but none has undergone characterization similar to BV-2 cells, and their use was quite limited. In general, earlier experiences have taught us that any cell line must first be shown to respond similarly to the primary cells they are to model before they are used in experiments.

Because species of cell lines is a fundamental and non-negligible issue, we wonder whether the mistake was just a slip of the pen. However, the result will not influence the conclusion, which inspired us to pursue a meticulous scientific attitude.