Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons

Abstract

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al.¹.

Keywords: cell biology; cell culture; cell isolation.

Graphical abstract

Figure 1

Schematic and gross images of pig spiral colon Pictures demonstrate: (A) obtaining a spiral colon specimen from the central flexure (related to Part 1, Step 3). (B–D) (B) trimming fat and mesentery away from the serosal surface (related to Part 1, Step 7), (C) orienting the colon specimen mucosa up (related to Part 1, Step 8), and (D) cutting and stretching the colon specimen to a 3 cm × 3 cm square (related to Part 1, Step 10). Created with BioRender.com.

Figure 2

Microdissection of the different layers of the colon Pictures illustrate the different steps mentioned in the protocol: (A) Schematics illustrate the steps of isolation detailed in A: mucosa (Muc), epithelium (Epi), lamina propria (LP), muscularis mucosa (MM), submucosa (SM), circular (CM) and longitudinal (LM) layers of the muscularis (Mus), and serosa (Ser). (B) Gross removal of the mucosa (related to Part 1, Step 11), isolation of the submucosa containing the submucosal plexus (SM) (related to Part 1, Step 12), and removal of the circular muscle to isolate the longitudinal muscle/myenteric plexus (LMMP) (related to Part 1, Step 15). (C) H&E staining confirms precision of dissection at 10X magnification (related to Part 1, Step 16). (D) H&E staining at 20X magnification (related to Part 1, Step 16). Scale bar for H&E, 100 μm. Created with BioRender.com.

Figure 3

Schematic depicts experimental design Created with BioRender.com.

Figure 4

Establishment and expansion of primary cultures of enteric glial cells Bright-field images of primary cultures of enteric glial cells isolated from neonatal and juvenile pig colon submucosa (SM) and longitudinal muscle/myenteric plexus (LMMP) on day in vitro 1 (DIV1) before and after medium change to illustrate changes in cellular density due to the removal of unadhered cells and debris, DIV3 to illustrate the beginning of the formation of enteric glial cell clusters, and DIV5 to illustrate the expansion of enteric glial cells and the formation of a glial network. Scale bar, 100 μm (related to Part 1, Steps 34–35).

Figure 5

Enteric glial cell primary cultures from the submucosa (SM) and longitudinal muscle/myenteric plexus (LMMP) on day 1 in vitro following the first passage (related to Part 2A, Steps 51–52) Scale bar, 50 μm.

Figure 6

Expression of the enteric glial cell markers GFAP and S-100β in primary cultures isolated from the submucosa and longitudinal muscle/myenteric plexus of neonatal and juvenile pig colons Primary cultures of enteric glial cells isolated from neonatal and juvenile pig colon submucosa (SM) and longitudinal muscle/myenteric plexus (LMMP) are >95% positive for the enteric glial markers GFAP and S-100β at passages 0 and 1 (related to Part 2C, Step 90). (A) Representative pictures of enteric glial cell primary cultures isolated from neonatal and juvenile pig colon SM and LMMP at passage 0 stained for the enteric glial cell markers GFAP (red) and S-100β (magenta). (B) Quantification of the average percentage of cells immunoreactive for the enteric glial cell markers GFAP and S-100β in SM and LMMP enteric glial cell primary cultures at passage 0. (C) Representative pictures of enteric glial cell primary cultures isolated from neonatal and juvenile pig colon SM and LMMP at passage 1 stained for the enteric glial cell markers GFAP (red) and S-100β (magenta). (D) Quantification of the average percentage of cells immunoreactive for the enteric glial cell markers GFAP and S-100β in SM and LMMP enteric glial cell primary cultures at passage 1. Data are expressed as mean ± SEM. No significant differences were identified by Two-way ANOVA Tukey’s multiple comparisons test (n = 3–4 independent experiments, at least 6 fields were analyzed per well). DAPI (blue) shows nuclei. Scale bar, 50 μm.

Figure 7

Expression of the enteric glial cell marker SOX10 in primary cultures isolated from the submucosa and longitudinal muscle/myenteric plexus of neonatal and juvenile pig colons Only a fraction of enteric glial cells in primary cultures isolated from neonatal and juvenile pig colon submucosa (SM) and longitudinal muscle/myenteric plexus (LMMP) are immunoreactive for SOX10 at passage 0 and 1 (related to Part 2C, Step 90). (A) Representative pictures of enteric glial cell primary cultures isolated from neonatal and juvenile pig colon SM and LMMP at passage 0 stained for the enteric glial cell marker SOX10 (green). (B) Quantification of the average percentage of cells immunoreactive for the enteric glial cell marker SOX10 in SM and LMMP enteric glial cell primary cultures at passage 0. (C) Representative pictures of enteric glial cell primary cultures isolated from neonatal and juvenile pig colon SM and LMMP at passage 1 stained for the enteric glial cell marker SOX10 (green). (D) Quantification of the average percentage of cells immunoreactive for the enteric glial cell marker SOX10 in SM and LMMP enteric glial cell primary cultures at passage 1. Data are expressed as mean ± SEM. No significant differences were identified by Two-way ANOVA Tukey’s multiple comparisons test (n = 3–4 independent experiments, at least 6 fields were analyzed per well). DAPI (blue) shows nuclei. Scale bar, 50 μm.

Figure 8

Purity analysis of P0 primary cultures of enteric glial cells isolated from the submucosa and longitudinal muscle/myenteric plexus of neonatal and juvenile pig colons Enteric glial cell primary cultures isolated from neonatal and juvenile pig colon submucosa (SM) and longitudinal muscle/myenteric plexus (LMMP) comprise less than 10% of cells immunoreactive for myofibroblast or neuronal markers at passage 0 (related to Part 2C, Step 90). (A) Representative images (whole well stitching) of enteric glial cell primary cultures isolated from neonatal and juvenile pig colon SM and LMMP at passage 0 immunolabeled for the pan-neuronal marker PGP9.5 (green), myofibroblast marker α-SMA (red), and glial cell marker S-100β (magenta). (B) Quantification of the average percentage of cells immunoreactive for the pan-neuronal marker PGP9.5 and myofibroblast marker α-SMA in SM and LMMP enteric glial cell primary cultures at passage 0. Scale bar, 1 mm. Data are expressed as mean ± SEM. No significant differences were identified by Two-way ANOVA Tukey’s multiple comparisons test (n = 3–4 independent experiments, at least 6 fields were analyzed per well). DAPI (white) shows nuclei. Scale bar, 1 mm.

Figure 9

Purity analysis of P1 primary cultures of enteric glial cells isolated from the submucosa and longitudinal muscle/myenteric plexus of neonatal and juvenile pig colons Enteric glial cell primary cultures isolated from neonatal and juvenile pig colon submucosa (SM) and longitudinal muscle/myenteric plexus (LMMP) comprise less than 10% of cells immunoreactive for myofibroblast or neuronal markers at passage 1 (related to Part 2C, Step 90). (A) Representative images (whole well stitching) of enteric glial cell primary cultures isolated from neonatal and juvenile pig colon SM and LMMP at passage 1 immunolabeled for the pan-neuronal marker PGP9.5 (green), myofibroblast marker α-SMA (red), and glial cell marker S-100β (magenta). (B) Quantification of the average percentage of cells immunoreactive for the pan-neuronal marker PGP9.5 and myofibroblast marker α-SMA in SM and LMMP enteric glial cell primary cultures at passage 1. Data are expressed as mean ± SEM. No significant differences were identified by Two-way ANOVA Tukey’s multiple comparisons test (n = 3–4 independent experiments, at least 6 fields were analyzed per well). DAPI (white) shows nuclei. Scale bar, 1 mm.