The Culture Dish Surface Influences the Phenotype and Dissociation Strategy in Distinct Mouse Macrophage Populations

Abstract

The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity of harvested macrophages. Several previous studies assessed the detachment efficacies of enzymatic and non-enzymatic methods for harvesting the single cell suspensions of macrophages, but a comprehensive study assessing different dissociation methods and culture conditions for detaching functionally different macrophage populations has not yet been reported. In this study,viathe well-established GM-CSF and M-CSF differentiated bone marrow derived macrophage models (GM-BMDMs and M-BMDMs), we compared four commonly used enzymatic (trypsin and accutase) and non-enzymatic (PBS and EDTA) dissociation methods along with necessary mechanical detaching steps (scraping and pipetting) to evaluate the viable cell recovery and cell surface marker integrality of GM-BMDMs and M-BMDMs cultured on standard cell culture dish (TC dish), or on culture dish (noTC dish) that was not conditioned to enhance adherence. The data showed that accutase yielded a better recovery of viable cells comparing with PBS and EDTA, especially for tightly adherent GM-BMDMs on TC dishes, with a relatively higher level of detected cell membrane marker F4/80 than trypsin. An additional gradient centrifugation-based dead cell removal approach could increase the proportion of viable cells for TC cultured GM-BMDMs after accutase dissociation. Furthermore, transcriptome analysis was performed to evaluate the putative influence of culture dishes. At steady state, BMDMs cultured on noTC dishes exhibited more proinflammatory gene expression signatures (e.g. IL6, CXCL2 and ILlβ) and functions (e.g. TNF and IL17 signaling pathways). Similar inflammatory responses were observed upon LPS challenge regardless of culture conditions and differentiation factors. However, in LPS treated samples, the difference of gene expression patterns, signaling pathways and molecular functions between TC and noTC cultured BMDMs were largely dependent on the types of growth factors (M-CSF and GM-CSF). This observation might provide valuable information forin vitromacrophage studies.

Integrated transcriptomic and metabolomic analysis reveals the metabolic programming of GM-CSF- and M-CSF- differentiated mouse macrophages

“…In contrast to their high expression in LPS/IFNg-polarized classically activated M1 macrophages, the gene expression levels of NOS family members were at low levels in both GM and M cells and no significant differences were observed. According to our previously published RNA-seq data (6), iNOS is a strong LPS-induced gene regardless of the differentiation/maturation with M-CSF or GM-CSF (FPKM value 0.17 vs 226.75 for M vs M_LPS_3h; FPKM value 0.15 vs 79.69 for GM vs GM_LPS_3h). In addition, LPS induced iNOS expression catalyzes the massive production of NO and consequently impaired the OXPHOS activity of macrophages (51,52).…”

“…Mouse BMDMs were prepared as previously described ( 6 ). Briefly, bone marrow cells were collected from femurs.…”

“…M-CSF is known for its role in inducing macrophage differentiation towards an antiinflammatory phenotype (M2-like macrophages), whereas GM-CSF induces macrophage differentiation towards a proinflammatory phenotype (M1-like macrophages) (3,4). In recent years, a number of studies have been reported in an effort to fully elucidate the various aspects of BMDMs (5)(6)(7). For example, the key chromatin modulators (e.g.…”

“…H3K27me3) ( 5 ) and essential signaling pathways (e.g. type I interferon signaling) ( 8 , 9 ) for macrophage differentiation, the influence of culture conditions on inflammatory responses ( 6 ), and the heterogeneity of macrophage populations have been thoroughly described in literature ( 10 ). To better understand macrophage plasticity, more attention has been placed on orchestrating the course of macrophage differentiation.…”