Notes
A Simple Method for Purified Primary Culture of Enteric Glial Cells from Mouse Small Intestine
2022 Volume 45 Issue 4 Pages 547-551
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2022 Volume 45 Issue 4 Pages 547-551
Enteric glial cells (EGCs) have been recognized as an important cell type constituting the enteric nervous system. EGCs control intestinal function and homeostasis through interactions with enteric neurons, epithelial cells and immune cells. To clarify the roles of EGCs in intestinal function and homeostasis, especially through secretion of and response to physiologically active substances, purified EGCs in primary culture have great advantages as an experimental tool. However, contamination by other cell types, fibroblasts in particular, is problematic in conventional primary myenteric culture. Previous methods to purify primary EGCs take a long time (over one month), are expensive, and are labor intensive. In the present study, we sought to purify primary EGCs from mouse small intestine by a simpler method than previous ones. After trying various protocols, we have established a method combining serum-free treatment and scraping fibroblasts off with a pipette tip. With our method, a purity of more than 90% EGCs was achieved after 14-d primary culture. Thus, our method is useful for investigating the roles of EGCs in intestinal function and homeostasis in detail in vitro.
Enteric glial cells (EGCs) have been recognized as an important cell type constituting the enteric nervous system. EGCs control intestinal function and homeostasis through interactions with enteric neurons, epithelial cells and immune cells.1–3) To clarify the roles of EGCs in intestinal function and homeostasis in detail, purified EGCs in primary culture have great advantages as an experimental tool. For example, using purified primary EGCs, the secretion of various substances from EGCs and the response of EGCs to stimulatory substances can be measured without interference from other cell types.4–6) In addition, primary cells are generally expected to retain the native properties of that cell type more than transformed cell lines.
However, contamination by other cell types, fibroblasts in particular, is problematic in conventional primary myenteric culture. Previously, Bannerman et al. and Rühl et al. used a method combining antimitotic agent treatment and antibody complement-mediated cytolysis to purify EGCs in primary culture.7,8) As an alternative method, Le Berre-Scoul et al. combined differential centrifugation and cloning.6) Although these methods achieved a purity of more than 95% EGCs, they take a long time (over one month), are expensive, and are labor intensive. These drawbacks have hampered extensive studies of EGCs in vitro.
In the present study, we sought to purify primary EGCs from mouse small intestine by a simpler method than previous ones. We tried various protocols including basal medium change, serum-free treatment and fibroblast scraping, and established our best method to obtain purified primary EGCs. Among various protocols we tried, a combination of serum-free treatment and scraping fibroblasts off with a pipette tip was the most effective. Our established method permitted a purity of more than 90% EGCs.
C57BL/6J mice were purchased from Japan SLC (Hamamatsu, Japan) and bred in Nagoya City University. Animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals revised 1996, and approved by the animal care and use committee at Nagoya City University.
The method of primary culture of the myenteric plexus was largely described previously.9) Muscle strips with the myenteric plexus were isolated in phosphate-buffered saline (PBS) by stripping the mucosa away from the small intestine of C57BL/6J mice on postnatal day 20–22 using fine forceps. After cutting them into pieces of approximately 2-mm length with scalpel blades, the tissues were incubated for 1 h at 37 °C in 2 mL of a digestion solution composed of Eagle’s basal medium with Hank’s salts containing 300 U/mL collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 20 U/mL deoxyribonuclease I (Sigma-Aldrich, St. Louis, MO, U.S.A.). After vortexing them for 15 s, the supernatant was collected and stored on ice. The undigested tissues were again subjected to incubation for 30 min in 1 mL of the digestion solution, vortexing, and collection of the supernatant. After the total supernatant was centrifuged (1100 rpm/220 × g, 2 min, 4 °C), the cell pellet was suspended in 3 mL of a serum-supplemented culture medium composed of Medium 199 (M199; Sigma-Aldrich) or Dulbecco’s modified eagle medium (DME)/F12 (Gibco/Thermo Fisher Scientific, Waltham, MA, U.S.A.) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 50 U/mL penicillin G potassium (Meiji Seika, Tokyo, Japan) and 100 µg/mL streptomycin sulfate (Meiji Seika). After centrifugation, the cell pellet was resuspended in 0.6 mL of the serum-supplemented culture medium and strained through a 70-µm nylon mesh filter (Falcon/Corning, Corning, NY, U.S.A.), which produced a cell suspension at a concentration of 1–3 × 105 cells per mL. Approximately 200 µL of the cell suspension was plated on a 35-mm culture dish (S35-DC12; Fine Plus International, Kyoto, Japan), the bottom of which was coated with poly-L-lysine (MW 30000–70000; Sigma-Aldrich). After 10-min incubation, 2 mL of the culture medium was added to each dish. The cells were incubated at 37 °C in 5% CO2/95% air. The culture medium was exchanged with a fresh one at 2, 6, 9 and 11 d in vitro (DIV).
To reduce the proliferation of fibroblasts, serum-free treatment was performed by culturing the cells in serum-free medium supplemented with G5 (Gibco/Thermo Fisher Scientific) and/or N2 (FUJIFILM Wako Pure Chemical Corporation) supplement in place of FBS during 2–6 DIV. Scraping of fibroblasts was performed at 2, 6, 9 and 11 DIV. We identified fibroblasts by their flat morphology under a phase contrast microscope (CK2; Olympus, Tokyo, Japan) and scraped them off with a 200-µL pipette tip (SATP-1002; Ikeda Scientific, Tokyo, Japan). In contrast, EGCs have a small cell body with long and thin processes.
ImmunocytochemistryThe cultured cells were fixed for 10 min in PBS containing 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation). After blocking for 60 min at room temperature in PBS containing 2.5% normal donkey serum (Chemicon/Merck Millipore, Darmstadt, Germany) and 0.3% Triton X-100, the fixed cells were incubated overnight at 4 °C in PBS containing a rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1 : 2400; 490740; Shandon, Pittsburgh, PA, U.S.A.) and a mouse anti-alpha smooth muscle actin (αSMA) antibody (1 : 1600; ab7817, Clone 1A4; Abcam, Cambridge, U.K.). Subsequently, they were incubated overnight at 4 °C in PBS containing a Cy2-conjugated donkey anti-rabbit immunoglobulin G (IgG) antibody (1 : 800; 711-225-152; Jackson ImmunoResearch, West Grove, PA, U.S.A.) and a Cy5-conjugated donkey anti-mouse IgG antibody (1 : 600; 715-175-151; Jackson ImmunoResearch), and then incubated for 30 min at room temperature in PBS containing 0.25 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Immunocytochemical images were acquired using a confocal laser scanning microscope (LSM800; Carl Zeiss, Jena, Germany) equipped with a 10 × objective (N.A. = 0.45; Plan-Apochromat; Carl Zeiss). For quantification, we manually counted the number of GFAP (for EGCs)-, αSMA (for fibroblasts)- or DAPI (for total cells)-positive cells in images of 640 × 640 µm.
At first, we performed primary myenteric culture from mouse small intestine according to one of the previous methods,10) in which M199 supplemented with 10% FBS was used as the culture medium (Fig. 1A; Basal medium = M199, Supplement during 2–6 DIV = FBS). However, fibroblasts (αSMA-positive cells) were a predominant cell population (68.1 ± 4.5%), whereas EGCs (GFAP-positive cells) were a small cell population (26.6 ± 4.1%) in these cultures at 14 DIV (Figs. 2A, 3, “M199, FBS, no scrape”). As the basal medium, DME/F12 is more often used than M199 in relatively recent methods for primary myenteric cultures from rodent intestine.6,11,12) Therefore, we changed the basal medium from M199 to DME/F12 (Fig. 1A; Basal medium = DME/F12, Supplement during 2–6 DIV = FBS). As a result, the total cell density was increased (Figs. 2B, 3, “DME/F12, FBS, no scrape”). Importantly, the EGC percentage was increased to 41.5 ± 2.4% and became nearly equivalent to the fibroblast percentage (51.3 ± 2.5%). Thus, DME/F12 was used as the basal medium in the subsequent study. However, EGC purity was still insufficient with this method.
(A) Schema showing our various protocols. First, we compared M199 and DME/F12 as the basal medium (green arrow). Second, we tried serum-free treatment with G5 and/or N2 supplement during 2–6 d in vitro (DIV) (orange arrow). Third, we tried scraping fibroblasts off with a pipette tip at 2, 6, 9 and 11 DIV (blue arrows). (B) An example of scraping of fibroblasts. Phase contrast images were captured before and after scraping of fibroblasts enclosed in the blue dotted ellipses. Fibroblasts have flat morphology, whereas EGCs have a small cell body with long and thin processes. Scale bar = 50 µm.
The cells were cultured by each method for 14 d and processed for immunocytochemistry against GFAP (green) and αSMA (red) and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; blue). The left labels show “basal medium, supplement during 2–6 DIV, with or without scrape.” Scale bar = 50 µm.
“Other cells” are GFAP- and αSMA-negative cells. n = 60 (M199, FBS, no scrape), 182 (DME/F12, FBS, no scrape), 109 (DME/F12, FBS, scrape), 226 (DME/F12, N2, scrape), 119 (DME/F12, G5, scrape) or 183 (DME/F12, G5 + N2, scrape) fields from 3–5 dishes of independent experiments. The total cell number we counted per condition was 6524 (M199, FBS, no scrape), 27429 (DME/F12, FBS, no scrape), 2638 (DME/F12, FBS, scrape), 17349 (DME/F12, N2, scrape), 11292 (DME/F12, G5, scrape) or 22802 (DME/F12, G5 + N2, scrape) cells. Error bars represent standard error of the mean (S.E.M.).
To increase EGC purity, we tested a cloning cylinder to isolate EGCs.6) However, the viability of the reseeded EGCs was reduced and fibroblast contamination was unavoidable by this method (data not shown). Therefore, we did not use a cloning cylinder any more.
Next, we tested scraping fibroblasts to remove them. We identified fibroblasts by their flat morphology under a phase contrast microscope and scraped them off with a pipette tip (Fig. 1B) at 2, 6, 9 and 11 DIV (Fig. 1A, “Scrape”). At 14 DIV with this method (Fig. 1A; Basal medium = DME/F12, Supplement during 2–6 DIV = FBS, Scrape), the EGC percentage was markedly increased to 67.0 ± 3.0% (cf. fibroblasts: 21.5 ± 2.7%), although cell density of total cells was severely decreased (Figs. 2C, 3, “DME/F12, FBS, scrape”).
Finally, we tested serum-free treatment. We intended to reduce the proliferation of fibroblasts by culturing in a serum-free medium during the early culture period (2–6 DIV). To prevent this treatment from reducing the proliferation of EGCs, G5 supplement, the supplement developed for serum-free culture of glial cells,13) was added to the culture medium (Fig. 1A; Basal medium = DME/F12, Supplement during 2–6 DIV = G5, Scrape). For comparison, N2 supplement, the supplement developed for serum-free culture of neurons,14) was also tested (Fig. 1A; Basal medium = DME/F12, Supplement during 2–6 DIV = N2, Scrape). As a result, the serum-free treatment with N2 supplement increased the total cell density (Figs. 2D, 3, “DME/F12, N2, scrape” vs. “DME/F12, FBS, scrape”). However, the percentages of EGCs (69.8 ± 1.9%) and fibroblasts (19.1 ± 1.7%) were not different from those under the serum-supplemented condition. In contrast, the serum-free treatment with G5 supplement effectively increased the density of EGCs, which resulted in a further increase in the EGC percentage to 87.4 ± 1.4% (cf. fibroblasts: 9.0 ± 1.2%), thus making EGCs a predominant cell population (Figs. 2E, 3, “DME/F12, G5, scrape”). Moreover, the EGC density was further increased when both G5 and N2 supplements were added to the serum-free culture medium (Figs. 2F, 3, “DME/F12, G5 + N2, scrape”). The EGC percentage reached 90.3 ± 0.9% (cf. fibroblasts: 6.3 ± 0.8%) with this method.
In this way, we succeeded in establishing a method to culture and obtain primary EGCs from mouse small intestine with a purity of more than 90%. The essential steps of this method are serum-free treatment during the early culture period and scraping fibroblasts off with a pipette tip. This method is simpler, cheaper, and takes less time (two weeks) to purify primary EGCs in comparison with several previous methods. Thus, this method is useful for investigating the roles of EGCs in intestinal function and homeostasis in detail in vitro.
In our established method, we used both G5 and N2 supplements as serum replacements during the early culture period (2–6 DIV). Gomes et al. also used DME with both G5 and N2 supplements for primary culture of embryonic mouse intestine and found that this medium increases the number of neurons compared with DME with either G5 or N2 supplement.15) However, they did not describe the effects of this medium on EGCs. Our study showed that this medium increases numbers of not only neurons but also EGCs. Further, it is noteworthy that G5 and N2 supplements were effective even when added only during the early culture period.
In our trials, the method using a cloning cylinder did not succeed. This may have been because this method requires cell harvesting and re-suspension, which reduce the viability of primary EGCs. In contrast, our established method does not require cell harvesting or re-suspension, maintaining cell viability. This is one of the advantages of our established method.
We thank the Research Equipment Sharing Center at Nagoya City University. This study was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Nos. 16K08744, 19K07485).
The authors declare no conflict of interest.
