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. 2011 Jul 16;32(1):67–75. doi: 10.1007/s10571-011-9735-9

A Much Convenient and Economical Method to Harvest a Great Number of Microglia

Kun Qin 1, Ye-Hai Li 1, Ge Tian 2, Wei-Wei Xu 1, Peng Li 1, Run Zhang 1, Zheng-Yang Li 1, Xiao-Dan Jiang 1,✉
PMCID: PMC11498607  PMID: 21833552

Abstract

Microglia, implicating in such neuro-pathologies as brain inflammation, neurodegeneration, glioma, and neurogenesis, play an important role in central nervous system. Advanced research on microglia is crucial in exploring the neuro-pathology and neuro-physiology of these diseases, so how to culture large numbers of microglia in vitro becomes the base of a research. The wildly used method, at present, obtaining microglia from murine cannot fulfill the requirement of research, costing too much time and needing too many rats. We intend to introduce an optimized method that can harvest large quantities of microglia with high purity. Neonatal 2–3 days old Wistar rats were sacrificed and the cerebral cortices were trypsinized. We primarily cultured mixed cortical cells for 8–10 days. The microglia were harvested from the liquid supernatant; the left cells in the mixed cortical glial culture were passaged at a 1:2 density. After another 8–10 days of culture, microglia were collected again. And then, we passaged the left cells again for acquiring microglia from the third collection. We did not add additional mitogens in the experiment. At last, on average, 7.0 × 106 microglia were collected from one neonatal rat. By this modified method, much more microglia can be effectively and easily harvested comparing with the usual protocol before. We compared the characteristics of microglia harvested from these three passages, such as morphology, phenotype, purity, and abilities on proliferation, secretion, and phagocytosis. The cells presented typical microglia morphology, having phenotype markers of CD11b/c and CD45. The microglia from these three passages retained similar phagocytosis and secretion functions. Expanded population of microglia for investigation can be provided by this easy method in a short time with little cost and few rats.

Keywords: Microglia, Glial culture, Lipopolysaccharide, CD11b/c, CD45

Introduction

Microglia, the resident macrophages of the central nervous system (CNS), are very important in the immune system of the brain with similar functions as macrophages (Landreth 2009; Yang et al. 2010). Microglia, a kind of glial cells derived from early embryonic myeloid-monocytic cells, are antigen presenting cells in the CNS (Cardona et al. 2006; Moussaud and Draheim 2010). The major functions of microglia within the brain are keeping homeostasis and promoting inflammation in the infected or damaged tissue (Kaur et al. 2010; Mariani and Kielian 2009; Nelson et al. 2002). It is found that microglia have a close relationship with neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (Perry et al. 2010; Ringheim and Conant 2004). Microglia not only eliminate bacteria, virus, or other infectious agents in meningitis and encephalopathies (Kaur et al. 2010; Mariani and Kielian 2009; Rock and Peterson 2006; Yang et al. 2010) but also play an important role in gliomagenesis (Ghosh and Chaudhuri 2010; Graeber et al. 2002; Watters et al. 2005). It is much clear that microglia play a critical role in neuroinflammation. Therefore, it is necessary to study the features of microglia in vitro and further understand the mechanism of pathophysiology in neuroinflammation in vivo. It is an important precondition to get large numbers of microglia efficiently and conveniently in vitro for further research. Several methods are employed to obtain microglia, including separation through gradient density centrifugation (Gingras et al. 2007; Moussaud and Draheim 2010), fluorescence-activated or magnetic-activated cell sorting (Ford et al. 1995; Marek et al. 2008), and isolation by mild trypsinization (Saura et al. 2003). Nevertheless, these methods are not easy to operate, costing much time and a larger population of animals are killed. Meanwhile, as microglia alone are difficult to survive very long in vitro, stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF), are used to promote the proliferation of microglia to acquire large numbers of microglia in vitro (Giulian and Ingeman 1988; Liva and de Vellis 2001; Ponomarev et al. 2005; Tomozawa et al. 1996). Although addition of exogenous mitogens during culture expands the population of microglia, it also results in additional expense on the experiment. The most frequently used protocol is to shake and separate them from the mixed glial cells reported by Giulian and Baker (1986), which has approved to be a reliable method. Highly enriched microglia could be obtained through this protocol; nonetheless, the major disadvantage of this method is hard to collect enough quantity of microglia for given study. To culture microglia rapidly, efficiently, economically, and conveniently in our study, we modified the procedure of mixed glial culture, which we used to isolate and acquire large numbers of highly purified microglia from the rat brain.

Experimental Procedure

Cell Culture

Neonatal 2–3 days old Wistar rats were killed; the cerebral hemispheres were dissected out; and the meninges, hippocampus, basal ganglion, and olfactory bulb were carefully removed with microsurgical instruments under microscope. The remaining cerebral cortices were washed, minced with scissors, and trypsinized. The cells were harvested, plated onto tissue culture T-75 flasks, and cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 containing 10% fetal bovine serum (FBS, Hyclone). After cultured for 8–10 days in vitro, microglia were collected through shaking the flasks and gently blowing with pipette. Microglia were calculated and cultured in T-25 flasks in the conditional medium (DMEM/F12 containing 10% FBS). The left cells in the mixed cortical glial culture were passaged at a 1:2 density and cultured with fresh DMEM/F12 containing 10% FBS, and then, microglia of the second generation were collected after another 8–10 days of culture. And microglia of the third generation were obtained through the same protocol. All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996 and Southern Medical University ethical principles.

Immunocytochemistry Analysis

Microglia were cultured in 24-well plate for 24 h, and then fixed with 4% paraformaldehyde for 30 min at room temperature, followed by immunostaining with such primary antibodies as mouse anti-rat CD11b/c which is equivalent antibody to OX-42 (1:200, Abcam,) and rabbit anti-rat CD45 (1:200, Santa Cruz). Secondary antibodies such as DyLightTM 488 goat anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratories) and DyLightTM 594 goat anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories) were used to detect the primary antibodies. Hoechst fluorescent 33342 (Sigma) were used to stain the nucleus. Each experiment was performed three to four times. The images of the cells were captured with the fluorescence microscope (LSM 510, Leica).

Flow Cytometry Analysis

Flow cytofluorometry was used to evaluate the phenotype and purity of microglia. The cells were harvested and resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). After being washed, the cells were incubated for 20 min with FITC-conjugated primary antibodies: anti-rat CD45 (1:200, Abcam), anti-rat CD11b/c (1:100, Abcam), and anti-rat CD86 (Biolegend, 0.5 μg:100 μl), or with phycoerythrin (PE) conjugated primary antibody: anti-rat CD80 (ebioscience, 0.5 μg:100 μl). We used chicken anti-rat glial fibrillary acidic protein (GFAP, 1:1,000, Abcam) as primary antibody to incubate for 1 h at 37°C, then secondary antibody of Texas Red conjugated rabbit anti-chicken IgG (1:500, Abcam) was added and incubated for 1 h at 37°C. The samples were analyzed on flow cytofluorometry (BD Company).

Cell Proliferation Assay

Microglia were seeded in 96-well culture plates at a density of 1.0 × 104 cells/well within fresh DMEM/F12 containing 10% FBS. Within 7 days of culture, cell growth was measured using water-soluble tetrazolium salt (WST-8) assay (Dojindo Company) each day: 10 μl WST-8 was added to each well, and the cells were incubated for 1 h at 37°C. The absorbance of the sample at 450 nm was measured in a multiwell spectrophotometer (Multiskan MK3, Thermo Scientific Company).

Enzyme-Linked Immunosorbent Assay

To assess the secretion of microglia, we cultured 1.0 × 105 cells in each well of 24-well culture plate with DMEM/F12 without serum for 24 h. Lipopolysaccharide (LPS, sigma) at the concentration of 2 μg/ml was added into half of these wells. After 24 h of culture, we collected the supernatant. The levels of cytokines IL-10, IL-6, and TGF-β were assessed using enzyme-linked immunosorbent assay (ELISA) (R&D Systems) following the manufacturer’s protocol. The absorption was measured at 450 nm in a multiwell spectrophotometer (Multiskan MK3, Thermo Scientific Company). All treatments were completed at least three times and the cytokine concentrations were calculated according to standard concentrations of recombinant IL-10, IL-6, and TGF-β. The data were expressed as mean ± SEM.

Phagocytosis Assay

After being isolated from mixed glial culture, microglia were plated onto culture dish with DMEM/F12 containing 10% FBS. LPS at the concentration of 2 μg/ml was added. After 24 h of culture, fluorescence-labeled latex beads (L3030, sigma) were added at the concentration of 4 μl/ml for 1 h at 37°C. Then, we washed them with PBS and fixed the cells with 4% paraformaldehyde. Then, we observed whether the microglia were able to phagocytose the beads and phagocytosis was increased after LPS stimulation under confocal microscope.

Atomic Force Microscopy and Scanning Electron Microscopy Analyses

Microglia were plated on glass chamber slides in six-well plates for 24 h at a density of 2 × 105 cells/ml. For atomic force microscopy (AFM) imaging, slices were taken out and washed with PBS, fixed with 1% paraformaldehyde solution in PBS for 20 min and washed with PBS again, and washed with distilled water and air-dried finally. The imaging was conducted with tapping mode in a normal atmospheric environment using SPM-9500J3 AFM (Shimadzu Company). The range of the scanning probe was 62.5 × 62.5 μm with a scanning rate of 1 Hz. The images were acquired with a 512 × 512 data point resolution.

For scanning electron microscope (SEM) measurement, cover slips were washed with PBS and fixed in a 3% solution of glutaraldehyde for 2 h. After being washed with PBS again, the samples were fixed for 2 h in 1% osmic acid. Then, cells were dehydrated through increasing concentrations of ethanol. Subsequently, the cells were dried through critical point drying. The dry specimens were sputter coated with platinum before performing examination in the microscope. Hitachi S-3000 N electron microscope was used for the examinations.

Statistical Analysis

All experiments were completed at least three times and data were expressed as mean ± SEM. Statistical analyses were completed using SPSS 13.0. One-way analysis of variance (ANOVA) was used to determine the significance between groups. Significance was determined at a level of P < 0.05.

Results

Microglia Cell Culture

In our initial study, we followed the protocol of Gliulian (Giulian and Baker 1986) culturing microglia from mixed cortical glial culture with its results indicating that the number of microglia collected was very limited and it is hard to culture the microglia in vitro for a long time without astrocyte monolayer. On the basis of mixed cortical glial culture, we introduced a new convenient method to collect microglia. We successfully obtained large numbers of microglia by this method and it was much easy to operate. First, we isolated the microglia by blowing with pipette gently after the primary mixed cortical glial culture for 8–10 days. We could collect about 2.2–2.5 × 106 cells from one neonatal rat. Then, we trypsinized and passaged the left cells in the T-75 flask at a 1:2 density, which were mainly astrocyte monolayer and a small number of microglia. After another 8–10 days of culture, we were able to collect 2.0–2.4 × 106 cells from the second collection. Following this, we trypsinized and passaged rest of the cells in the T-75 flask at a 1:2 density again. After another 8–10 days, we could collect 2.0–2.7 × 106 cells from the third collection. We just did this cycle for twice. Finally, we successfully collected large quantities of microglia. For each collection (no matter it is the first, second, or third collection), we could obtain more than 2.0 × 106 cells. We obtained about 7.0 × 106 cells from only one of killed neonatal rat. On the other hand, we also significantly decreased the number of neonatal rats used in the experiment.

After being pictured through optical microscope, atomic force microscope, and scanning electron microscope, microglia were successfully harvested. We could see that the microglia showed the typical characteristic as amoeboid or ramified (Fig. 1). The morphology of microglia collected three times did not show any change. All the microglia exhibited either bipolar, multipolar, or rounded appearance. As viewed using different microscopes (Fig. 1), some of the microglia processed short and slender projections on the surface of cells, whereas others showed the long ones. Many vacuoles can be found in the cytoplasm in the microglia as well. These morphology characters found in our study were similar to the description by Giulian (Giulian and Baker 1986).

Fig. 1.

Fig. 1

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The cells isolated from whatever primary, second, or third collection according to the method in this study showed the typical morphology of microglia. The morphology of microglia under optical microscope (a), atomic force microscope (b), and scanning electron microscope (c) displayed the bipolar, multipolar, or rounded appearance with foot processes and some invaginations. Scale bars: a = 50 μm, b = 20 μm, and c = 10 μm

Microglial Cells From the First to Third Collection Having the Same Cellular Phenotype

We compared the phenotypes among three different collections with immunocytochemical staining and flow cytometry. The cells harvested from the three collections all contained high-purity CD11b/c and CD45-positive cells, and there were not significant differences among three collections on purity (Fig. 3). Meanwhile, under the fluorescence microscope, microglia, having similar morphologies, expressed CD11b/c and CD45 antibodies positively (Fig. 2).

Fig. 3.

Fig. 3

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Flow cytometric assay were used to examine the phenotypes of microglia. The microglia highly expressed CD11b/c (a) and CD45 (b) with its proportion of reaching higher than 95%. And microglia expressed much low levels of GFAP (c), CD80 (d), and CD86 (e). The bar chart showed the average proportion of the phenotype of microglia (n = 3)

Fig. 2.

Fig. 2

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The phenotype markers (CD11b/c and CD45) of the microglia showed no significant difference among three passages. Immuno-fluorescence pictures of microglia displayed that all microglia (from the first to third passage) positively expressed CD11b/c (left) and CD45 (right). The cellular nuclei were counterstained with Hoechst fluorescent 33342. BarCD11b/c = 25 μm, BarCD45 = 50 μm

Microglia Enjoying the Same Proliferating Situation

Microglia harvested from mixed cortical glial culture were plated in 96-well plates. Each well contained 1.0 × 104 cells. WST-8 assay was employed for 7 days to assess the proliferation rate of cells from the three collections to observe whether the cells had different ability in proliferation after being passaged. The line chart demonstrated that the cells from the three collections did not significantly proliferate within 1-week culture in vitro (Fig. 4).

Fig. 4.

Fig. 4

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Accounting for neonatal rat microglia from each collection (including the first, second, or third collection) after being cultured were more than 2.0 × 106 cells (b). There were not significant differences on the proliferation of microglia between the three collections (P > 0.05) (a). All microglia secreted a mass of cytokines (IL-6, IL-10, and TNF-α) after being treated with LPS (c). There were not significant differences on the secretion of cytokines between microglia harvested from the three collections (P > 0.05)

The Cytokines Secreted by Microglial Cells

The ability of producing cytokines was one of most important characters of microglia. We evaluated the amount of cytokines, such as TNF-α, IL-10, and IL-6 in supernatant secreted by the microglia isolated by this modified method. The microglia were separated into two groups. In group one, microglia were stimulated by LPS, and another one was negative control. We observed that microglia collected after passaged from the first or third collection had similar ability of secreting cytokines as compared with primary microglia after elicited by LPS (Fig. 4).

The Phagocytic Capacity of Microglial Cells

After blown with pipette, microglia were collected from mixed glial cells and cultured in fresh medium for 24 h. Then, latex beads were added to evaluate the phagocytosis of microglia. The phagocytic capacity of microglial cells on latex beads was observed under laser confocal microscope after 1 h. We could see that the red fluorescent latex beads were in the microglia (Fig. 5). After exposing to LPS, microglia phagocytized much more latex beads as compared with the negative controls (Fig. 5). And the microglia obtained from the three collections all had similarly phagocytic capacity.

Fig. 5.

Fig. 5

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Phagocytosis of microglia showed in picture a and b. Microglia treated by LPS (a) phagocytosed more fluorescent latex beads (arrow) than the one not stimulated (b). Scale bars = 10 μm

Discussion

In our study, we use a new method based on a mixed glial cell culture to enrich the population of microglia. This method is easy to use and requires no additional cytokines to generate sufficient numbers of microglia. Our key modification of the existing method is passaging of the remaining mixed glial cells. After many attempts, we found that when we passaged the remaining cells after isolation, the number of microglia was almost as great as that generated by primary cultures. Because of the convenient and economic collection of microglia that can be performed this way, this method is very helpful for the further study of microglia in vitro. Using this process, we are able to provide sufficient cells for subsequent research on microglia.

To confirm that the microglia generated by our method maintain the features of typical microglia, we compared the characteristics of microglia, including morphology, purity, and phenotype and capacity for proliferation, secretion, and phagocytosis. Using pictures taken on an optical microscope, an atomic force microscope, and a scanning electron microscope (Fig. 1), we found that microglia generated from secondary and tertiary mixed glial cell cultures were the same as the primary ones. The first collection was carried out under the classical method. As described previously, microglia exhibit their typical morphology with elongated bipolar, multipolar, or unipolar cells and amoeboid cells (Yang et al. 2010). The antigens CD11b/c and CD45 are the surface markers of microglia. We used anti-CD11b/c and anti-CD45 antibodies with flow cytometry to test the purity of isolated microglia from our three collections. Our data demonstrate that we were able to successfully harvest high-purity microglia, and there was no difference in the purity of microglia obtained from primary cultures, or from second or third passage mixed glial cell cultures (Fig. 3). To ensure that the microglia had the same functional capacity, we compared the secretory and phagocytic behavior of the collected microglia. The microglia obtained from passaged mixed glial cell cultures were able to secrete cytokines and phagocytose latex beads to the same extent as the initial cultures (Fig. 5).

The reasons why passaged mixed glial cells maintain the ability to proliferate and form a large number of microglia could be as follows. Many studies have assumed that, in a mixed glial cell culture, the proliferation of microglia depends on the glial cells that secrete the necessary factors to enable the microglia to survive and proliferate. However, because of contact inhibition, the glial cells are likely to be quiescent and not secrete many cytokines, if too many cells are present in the culture flask. Passaging then provides a stimulus that forces the glial cells to become active and to proliferate. The proliferation of glial cells then improves the proliferation of microglia. Additionally, interaction between microglia and astrocytes might be helpful in maintaining the proliferation of microglia in mixed culture. Finally, the mixed glial cell population contains many neural stem cells. Passaging stimulates the neural stem cells to differentiate and also promote proliferation of the microglia.

Compared with the typical methods used, our study has three main advantages. First, we were able to generate more glial cells from fewer neonatal rats using the method we describe; we collected about three times as many microglia from one neonatal rat, compared with the regular method. We acquired approximately 2.2–2.5 × 106 microglia from one neonatal rat using the mixed glial cell culture method commonly used. Using our modified process, we acquired approximately 7.0 × 106 microglia in total from one neonatal rat. In addition, our method simplifies the process of producing glial cells. The method introduced by Floden and Combs (2007) was to repeatedly collect the microglia from primary cultures of mixed glial cells, which was easy to perform. However, Floden also reported that the microglia obtained in second third time were far fewer than the first time. Instead, we just passaged the entire mixed glial cell culture. For each collection, we shook the flasks by hand and gently blew with a pipette. This was enough to harvest the desired number of microglia. The remaining microglia in the mixed glial cell culture could then be used as seed for the next collection, following passage. From the flow cytometric analysis, we were able to show that the purity of the microglia was the same at each collection. Last but not least, this method reduces the experimental cost and shortens the cycle of the experiment. Marshall and colleagues showed that the method they used could expand massive microglia from the subventricular zone (SVZ) of the mouse (Marshall et al. 2008). However, they had to first add many cytokines, such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), to amplify the cells obtained from the SVZ in vitro, before isolating microglia. Until then, microglia could undergo massive expansion through passaging. In addition, some studies reported that mitogenic factors, such as GM-CSF and M-CSF, could be added to the medium to maintain microglia in culture (Giulian and Ingeman 1988; Ponomarev et al. 2005; Tomozawa et al. 1996). In our experiment, we did not add any cytokines to stimulate the production of microglia. If additional mitogenic factors were added, we could harvest even larger numbers of microglia from such a mixed culture. Nevertheless, in this work, we aimed to report a more economical protocol to collect sufficient microglia for use in experiments. Therefore, using the protocol we present, no additional cytokines are required to collect a large quantity of microglia at a high purity. The number of microglia obtained from the second and third collection is the same as from the first one. This is extremely important in decreasing the cost of an experiment.

Although our work demonstrates that isolating large numbers of high-purity microglia with our method is practicable, some limitations still exist. It is possible that the phenotypes of microglia may still differ between the three collections, although we did not find such differences. It is likely that the passaged cells undergo some phenotypic change. It is possible that the astrocytes in mixed culture could secrete cytokines, such as GM-CSF or M-CSF, which could affect the characteristics of microglia. However, from the results of our work, we did not find that the microglia obtained from different collections had any significant difference.

In summary, we suggest that the protocol introduced here is very useful. It optimizes the often-used mixed glial cell culture to harvest microglia, which could provide large numbers of high-purity microglia for experiments. In addition, fewer neonatal rats need to be killed for research. The use of additional cytokines, such as GM-CSF, M-CSF, EGF, and bFGF, is not required, and most importantly, the method is very simple to carry out.

Acknowledgments

This research was supported by Funds for Key Sci-Tech Research Projects of Guangdong Province [YUECAIJIAO (2008) 258-2008A030201019, YUE KEJIBAN (2007) 05/06-7005206] and Funds for Key Sci-Tech Research Projects of Guangzhou [SUIKETIAOZI (2008)3-2008A1-E4011-6, 09B52120112-2009J1-C418-2] to Prof. Xiaodan Jiang.

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