Growth and Survival of Satellite Glial Cells in Different Culture Supplementations

Background: In dorsal root ganglion (DRG), satellite glial cells (SGCs) tightly surrounded neurons and modulated microenvironment and sensory transmission. However, the biological properties of primary SGCs in culture were not fully understood. In the present study, we provided a method to harvest abundant and high-purity SGCs from neonatal rats. Three supplementations containing Dulbecco ’ s Modified Eagle Medium (DMEM)/F12, DMEM high glucose (HG) and Neurobasal-A (NB) were used to evaluate SGCs growth and survival in culture. Results: CCK-8 proliferation assay showed the increased proliferation in DMEM/F12 and DMEM/HG, but not in NB medium. NB medium caused cell death indicated by Bax, AnnexinV and PI staining. Glutamine was the major nutrition source for SGCs in culture and its exogenous application improved the poor proliferation and severe cell death in NB medium. SGCs markers GS and GFAP were similar in three supplementations and intensively expressed in culture. Differently, GS but not GFAP was remarkable in the intact DRG under normal condition. Conclusions: These results suggested that SGCs growth in culture depended on time and culture supplementation and DMEM/F12 medium was recommended to get high-purity SGCs. Glutamine was the major nutrition and the key molecule to maintain cell growth and survival in culture. Our study shed a new light on understanding the biological property and modulation of glial cells in the peripheral sensory ganglia. In the present study, we provided a method to obtain high-purity SGCs culture from DRG of neonatal rats. We compared the biological properties of SGCs in DMEM/F12, DMEM/HG and NB supplementations and found that DMEM/F12 medium was valuable for SGC growth and survival. The poor proliferation and severe cell death in NB medium were improved by exogenous glutamine application. SGCs intensively expressed GFAP and GS in culture condition, whereas GS was a better SGC marker in the intact DRG under normal condition. Our study shed a new light on understanding the biological property and modulation of glial cells in peripheral sensory ganglia.


Introduction
In dorsal root ganglion (DRG), satellite glial cells (SGCs) were interconnected by gap junctions and tightly surrounded neurons [1,2]. SGCs modulated the neuronal microenvironment and sensory transmission [3,4]. Generally, SGCs in the peripheral nervous system were roughly equivalent in function to astrocytes in the central nervous system and contribute to pain [5]. It was widely reported that peripheral nerve injury and inflammation would cause SGCs changes in cell number and inflammatory mediators such as glutamate, adenosine triphosphate (ATP), gamma-amino-butyric acid (GABA), substance P, CGRP [6,7,8,9].
Till now, it was still a challenge to harvest high-purity SGCs. The primary DRG cell cultures mostly consisted of neurons, SGCs and a moderate number of macrophages [10,11,12]. Even in the neuronal culture, DRG neurons were contacted a coterie of adherent satellite cells not removed during the harvesting procedure and neurons without satellite cells were uncommon [13]. To purify glia in the primary culture, DMEM/F12 medium and cytosine arabinoside were used [14,15]. Sometimes, cytosine arabinoside was added to remove dividing cells after getting single-cell suspension and glial cell-derived neurotrophic factor were followed to harvest SGCs [16]. Recently, a novel primary culture method for obtaining high-purity satellite glial cells migrated from DRG explants was reported [17]. In this method, glia started to migrate from DRGs in 3 days, formed clusters after 7 days, and was to be sufficient for subculture after 14 days. Although enzymatic digestion procedure was not necessary in the migration method, it really took a very long time (more than 14 days) to get high-purity SGCs. The major concerns were how to trigger, control and promote the efficiency of SGCs migration from ganglia in the early stage. It also should be noted that SGCs might be undergoing phenotypic changes over time in culture [18]. Contrast to the well-established primary neuronal culture, it was still an interesting question on how to efficiently get high-purity SGCs from the peripheral sensory ganglia.
Local tissue inflammation and chemotherapeutic agent also caused SGCs activation [24,25]. GFAP was recently assessed to evaluate SGCs differentiation in cell culture condition [26].Collectively, GFAP was widely known as a marker of SGCs activation [27]. GS could catalyze adenosine triphosphate-dependent amidation of glutamate to glutamine and was known as a better marker for SGCs [28]. Tongtako et al found that SGCs in DRG were immunopositive for GFAP in dogs and monkeys, whereas GFAP in murine SGCs was absence. GS represented a good SGC marker in murine and simian but not in canine. The authors further showed an intermediate glial cell population with phenotypical characteristics of oligodendrocytes and astrocytes in SGCs [26]. These results strongly suggested that the expressions of SGCs markers were changeable. There was very limited data to compare the expression of SGCs markers in vitro and in vivo.
In the present study, we provided a method to obtain high-purity SGCs culture from DRG of neonatal rats. We compared the biological properties of SGCs in DMEM/F12, DMEM/HG and NB supplementations and found that DMEM/F12 medium was valuable for SGC growth and survival. The poor proliferation and severe cell death in NB medium were improved by exogenous glutamine application. SGCs intensively expressed GFAP and GS in culture condition, whereas GS was a better SGC marker in the intact DRG under normal condition. Our study shed a new light on understanding the biological property and modulation of glial cells in peripheral sensory ganglia. Unless otherwise stated, chemicals were purchased from Sigma-Aldrich. Antibodies, reagents and Kits were listed in Table S1 for details.

Isolation and culture of satellite glial cells
DRGs were enzymatically digested, dissociated as previously described [12].
Postnatal day 1-3 rats were killed by cervical dislocation and the spinal column was opened. Up to 10 DRGs along the vertebral column were removed under aseptic conditions and put into Petri dishes filled with cold, oxygenated Dulbecco's modified Eagle's medium(DMEM) (HyClone). The epineurium and nerve roots were removed and the DRGs were transferred to 5 mg/ml collagenase type Ⅱ (HyClone). The isolated DRGs were incubated with collagenase at 37°C for 20 min. Then DRGs was treated with 0.125% trypsin (HyClone) and 10 mg/ml DNase I (Roche) for 10 min at 37°C. The solution was spun at 1500 rpm for 5 min and enzyme was removed. After washing with fresh culture solution, pipetting ten times, DRGs were naturally precipitated for 5 minutes. Collect the supernatant, and centrifuged at 1500 rpm. Cells were resuspended with 10% fetal bovine serum and 1% penicillin/streptomycin and  Table 1.

Immunocytochemistry and immunohistochemistry staining
Immunocytochemistry was performed as previously described [29]. SGC cells attached to coverslips were fixed, permeabilized and blocked. Then coverslips were incubated with primary antibodies overnight at 4 °C . Then coverslips were incubated with the secondary antibodies for 2 h at room temperature. Immunohistochemistry staining DRG sections was performed as previously described [30,31]. The rats were anesthetized with i.p. pentobarbital (50 mg/kg) and fixed with 4% paraformaldehyde.
Tissues were dissected, postfixed for 8 h, and cryoprotected in 20% sucrose in PBS overnight at 4 °C. Transverse frozen sections (20 m thick) were cut on CM1900 freezing microtome (Leica, Germany), incubated for 4 h in 0.05% Triton X-100 and 10% goat serum in phosphate buffered saline (PBS) at room temperature, followed by incubation with primary antibodies at 4 °C overnight with agitation. After three washes with PBS, the sections were incubated with secondary antibodies for 2 h at room temperature. In the present study, we used the following primary antibodies (see Table S1

Cell proliferation assay
The proliferation rate of SGCs supplemented with DMEM/F12, DMEM/HG, and NB was examined by CCK-8 as previously report [32]. Briefly, SGCs were seeded in to 96-well culture plate (Corning) and maintained for different time points including 1, 2, 9 3, 5, 8, 10 days. Cell counting Kit-8 (CCK-8) solution (10 ml per well) was added at 37 º C for 1 h. Then, the optical density (OD) was measured at 450 nm wavelength using a microplate reader. Additionally, SGCs were imaged and counted by live cell imaging system (Cell-R, Olympus, Japan).

Cell death assay
In the present study, to test the death level of SGCs, cell death promotor Bax was detected by immunocytochemistry staining of Bax antibody (Santa Cruz). The method was performed as mentioned above. SGCs were seeded in a 24 well plate and cultured in DMEM/F12, DMEM/HG and NB for 8 days. DAPI nuclear counterstain was performed to reveal the percentage of Bax-positive cells.

Apoptosis was measured by Annexin V-PI Apoptosis Detection Kit (BestBio)
according to the manufacturer's instructions. Briefly, SGCs were incubated with distinct culture supplementations for 8 days. Cells were then incubated with Annexin V and propidium iodide (PI) buffer for 15 min at room temperature. Apoptotic cells were observed after staining with annexin V-FITC and PI and further imaged by fluorescence microscopy (Olympus). The percentages of Annexin V-and PI-positive cells were measured for quantitation.

Statistical analysis
All data were expressed as means ± SEM. Statistical analyses were performed using Prism 6 (v6.0e, GraphPad). For comparison between two groups, unpaired two-tailed t test was used. For the comparison between three or more groups, one-way ANOVA with Tukey post hoc analysis was performed. Normality and equal variance tests were performed for all statistical analyses. P <0.05 was considered statistically significant.

SGCs growth depended on time and culture supplementation and NB medium impaired cell proliferation
To obtain high-purify satellite glial cells (SGCs), following procedures were To test the effect of culture medium on the growth of SGCs, we used three supplementations containing DMEM/F12, DMEM/HG and Neurobasal-A (NB).
Firstly, we counted the number of SGCs under 100 times magnification. In DMEM/F12, the number of SGCs gradually increased with the prolonged culture time from 4 days to 8 days. Differently, the numbers of SGCs were not significantly changed in DMEM/HG and NB at 2 and 4 days (Fig. 1a). These results suggested that SGCs growth correlated with cell culture supplementation. Then, SGCs proliferation was evaluated by CCK-8 assay at a series of time points at 1, 2, 3, 5, 8 and 10 days after seeding. In DMEM/F12, optical density (OD) value dramatically increased after 5 days (0.3±0.01, n=5 reduplicates) and peaked at 10 days (0.9±0.04, n=5 reduplicates). DMEM/HG increased OD value at 10 days after seeding (0.6±0.07, n=5 reduplicates). However, OD value was not changed by NB medium even at 10 days after seeding (0.1±0.08, n=5 reduplicates) (Fig. 1b). We further observed the morphological characteristics of primary SGCs culture in DMEM/F12 medium under phase-contrast micrographs at 1, 2, 3, 5, 8 and 10 days. Except for the gradually increased number of cells, SGCs exhibited remarkable morphological changes from the early elliptical cells with full bodies to the late dipolar cells with protrusion (Fig.   1c). These results showed that SGCs growth depended on time and culture media.
DMEM/F12 was highly recommended to get abundant SGCs in several days, whereas NB medium impaired SGCs proliferation in culture.

SGC markers were not affected by the different culture supplementations
Then we assessed the effects of different supplementations on SGCs markers by immunofluorescent staining at 10 days. Both GFAP and GS were intensively expressed in the majority of SGCs in the different culture supplementations (Fig. 2a).
Expressions of SGCs markers in the majority of cells supported that our protocol was valuable to obtain high-purity SGCs. Additionally, our results suggested that SGC markers GFAP and GS were not significantly affected by culture supplementations.

NB medium caused severe cell death and apoptosis in SGCs
To characterize the influence of culture medium on SGC survival, cell death promotor Bax (Bcl-2 Associated X protein) was detected at 8 days. In DMEM/F12 and DMEM/HG medium, Bax expressions were rarely observed. In contrast, the majority of SGCs in NB medium were Bax-positive (Fig. 3a).  (Fig. 4c, n=6-8, p<0.05 or p<0.001). These results confirmed that NB supplementation caused the expressions of apoptosis markers and triggered cell death. Interestingly, Annexin V, PI and Bax could be shown in different SGCs, further supporting the different stages of cell death (Fig. S2). Collectively, these results suggested that NB medium caused severe cell death in culture condition.

Glutamine was the major nutrition source for SGCs in culture and its exogenous application improved cell growth and survival
To reveal the key regulator determining SGCs growth and survival in culture, we further checked the components of culture medium (Table 1) Interestingly, we found that the other major nutrition source L-glutamine was over 2.5 mM in DMEM/F12 and DMEM/FHG, whereas it was absence in NB medium.
GlutaMAX (L-alanyl-L-glutamine) was a dipeptide substitute for L-glutamine, maintained a fresh supply of L-glutamine during long-term culture and could not spontaneously break down to form ammonia. Therefore, we firstly used exogenous GlutaMAX into NB medium to test the roles of glutamine on SGCs growth (Fig. 5a) (Fig. 5b, n=6, p<0.001). These data demonstrated that exogenous glutamine application in NB medium increased SGCs proliferation in a dose-related manner.

GS, but not GFAP, was good SGC marker in vivo and in vitro under normal condition
Finally, we observed the expression profiles of SGC markers in the intact DRG (Fig.   S3). Immunofluorescent labeling showed that GS was detectable whereas GFAP was

Discussion
In the present study, three cell culture supplementations with DMEM/F12, DMEM/HG and NB were evaluated for SGCs growth and survival. We found that DMEM/F12 promoted SGCs growth whereas NB medium caused cell death indicated by Bax, AnnexinV and PI staining. As the major nutrition source for SGCs in culture, glutamine exogenous application improved the poor proliferation and severe cell death in NB medium. Our findings indicated that DMEM/F12 medium was recommended to get high-purity SGCs and glutamine will inhibit cell death and is the key molecule to maintain cell growth and survival in culture.
In the present study, we found the difference on the expression profiles of SGCs marker GFAP in culture condition and in the intact DRG. GFAP-and GS-positive SGCs were over 99%, 98% and 84% in DMEM/F12, high glucose and NB medium.
In the intact DRGs (P2 and P90), GFAP expression was nearly undetectable and GS-positive SGCs were tightly surrounded medium-and large-sized DRG neurons.
Our observations were consistent with the previous reports on the low expression of GFAP in normal DRGs [23,33,34]. GFAP was widely known as a marker of SGCs activation [27] and the strong GFAP staining in normal SGCs only reported in 4-month-old rodent [35,36] to the control medium [26]. It was also reported that the expression of GS decreased while purinergic receptor P2X7 was maintained over time in culture, indicating that SGCs might be undergoing phenotypic changes [18]. In our study, we found that GFAP and GS were similar in DMEM/F12, DMEM/HG or NB supplementation.
These results suggested that the expressions of SGC markers GFAP and GS were not significantly affected by culture supplementations. Nevertheless, it was still an interesting to investigate the phenotype switch of SGC markers, if true, in long-term culture condition and even in vivo.
The balance of cell death promotor Bax and repressor Bcl-2 contributed to cell survival [37,38]. In the peripheral sensory system, cell death was widely investigated in neurons and multiple chemicals could trigger the process. For example, cisplatin initiated the mitochondrial stress pathway in DRG neurons and NGF blocked death upstream of Bax activation [39]. Halothane increased neuronal cell death vulnerability by downregulating miR-214 and upregulating Bax [40]. Tunicamycin induced apoptosis through caspase activation and mitochondrial dysfunction in DRG neurons [41]. It was shown that Bax was required for neuronal apoptosis and permitted DRG neurons to survive in the absence of neurotrophin signaling. Bax knockout rescued programmed cell death and produced a 50% increase in the number DRG neurons [42,43,44]. Apoptosis or programmed cell death involved in a series of changes in morphological and biochemical properties. Annexin V, a 36-kDa calcium-binding protein associated with phosphatidylserine, was detectable in apoptotic cells and necrotic cells. Propidium iodide (PI) does not stain live or early apoptotic cells due to the presence of an intact plasma membrane. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases and PI passed through the membranes, intercalated into nucleic acids, and displayed red fluorescence. Therefore, the Annexin V/PI approach indicated the different stages of cell death [45,46].
To our knowledge, the mechanism of SGCs death was quite limited. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), it was shown that sciatic nerve injury induced apoptosis in SGCs, but not DRG neurons 30 days after injury [47]. NO-cGMP pathway also involved in cell death in DRG and blockade of NO or the cGMP synthesizing enzyme soluble guanylate cyclase, could result in apoptosis of neurons and Schwann cells [48]. Recently, it was shown that the number of caspase-3+ SGCs in culture was significantly reduced by fibroblast growth factor 2 (FGF-2), epidermal growth factor, (EGF), ciliary neurotrophic factor (CNTF), heregulin 1(HRG), or forskolin (Fors) supplementation [26]. These results suggested that multiple regulators contribute to the process of SGC apoptosis. In the present study, SGCs survival was evaluated by apoptosis makers including Bax, AnnexinV and propidium iodide (PI) in the different culture supplementations. We found that NB medium caused the dramatic increase in these apoptosis makers.
Interestingly, Bax, AnnexinV and PI could be detected in different cells, suggesting the different stages of SGCs apoptosis in culture.
Bax-related apoptosis prevention was verified in some reports on neuronal rescue strategies. For example, Isoquercetin ameliorated tunicamycin-induced apoptosis in rat DRG neurons via suppressing ROS-dependent endoplasmic reticulum stress [41].
Electro-acupuncture treatment resulted in a significant downregulation of Bax protein and its mRNA, but an upregulation of Bcl-2 after SCI [49]. Liu et al showed that Electro-acupuncture-modulated miR-214 prevented neuronal apoptosis by targeting Bax in rats after spinal cord injury [50]. In the present study, we firstly reported that exogenous glutamine improved the poor proliferation, reversed Bax, AnnexinV and PI expressions, and rescued the severe cell death in SGCs culture. Therefore, glutamine might become an attractive molecule to regulate Bax-related growth and survival in SGCs.
Glutamine and glucose are two major nutrition sources contributed to multiple biosynthetic pathways and cellular functions. Glutamine metabolism involved a glucose-independent tricarboxylic acid (TCA) cycle and supported cell survival and proliferation under hypoxia and glucose deficiency in cancer [51,52]. The entry of glutamine, after its conversion to glutamate and then to α-ketogslutarate, into the TCA and oxidation to succinate, fumarate, and malate were highly facilitated by MYC expression [53]. Glutamine also played important roles in immune system as an essential nutrient for lymphocyte proliferation and cytokine production, macrophage phagocytic plus secretory activities, and neutrophil bacterial killing [54,55]. Today, glutamine's metabolic and non-metabolic functions in cancer cell were recognized 51 .
In the present study, we found the poor proliferation and severe SGCs death in NB medium without glutamine. We also showed the protective effects of glutamine, but not glucose, on SGCs growth and survival. Our study supported that glutamine, but not glucose, was the key nutrition source for SGCs in culture. Glutamine was a multifaceted amino acid and promoted protein synthesis not only by delivering substrate and energy but also by stimulating transcription of genes in the promoter region [56]. It will be interesting to reveal the glutamine-activated singling pathway in SGCs growth and survival in future.

Conclusions
In summary, we demonstrated that SGCs growth in culture depended on time and culture supplementation and DMEM/F12 medium was recommended to get high-purity SGCs. Glutamine was the major nutrition source and its exogenous application improved growth and survival of SGCs in culture. The high-purity SGCs will be valuable to investigate the function and modulation of glia in the peripheral sensory nervous system.

Conflict of interest
The authors declare no competing financial interests.

Acknowledgements
Not applicable.

Authors' contributions
Author YQY conceived and designed the work. NW, YPL and RRW conducted experiments, XLW and YY managed cell culture, HW and TH performed data analysis, YQY and HW wrote the manuscript. All authors have read and approved the manuscript.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Ethics approval and consent to participate
All animal experiments were performed in accordance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of FMMU. The number of animals used and their sufferings were minimized. 21

Consent for publication
Not applicable.