Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

Summary

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

Abstract

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of >95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Introduction

Interest in the function of enteric glial cells (EGCs) has steadily increased due to their recognized roles in the gut integrity and homeostasis^1,2. In addition, EGCs vary according to their location along the length of the GI tract^3,4. EGCs release various trophic factors including glial cell-derived neurotrophic factor (GDNF), contribute to gut motility^1,5 and respond to microbial byproducts^6,7. Studies have indicated that the EGC pop....

Protocol

All animal experiments described were approved by the University of Michigan's Committee on the Use and Care of Animals.

1. Preparation of Sterile Poly-D-lysine (PDL) and Laminin Solutions

1. At least one day prior to cell isolation, prepare poly-D-lysine (PDL) and laminin coated plates.
   NOTE: Both 6-well and 12-well plates were prepared depending on the experimental goals. Typically, 12-well plates were used for the quantitative analysis using western blots; whereas, 6-well plates were used to hold autoclaved (sterile) coverslips for immunohistochemistry. 24-well plates were used to culture EGCs for Ca²⁺ flux....

Results

Preps were considered unsuccessful if GFAP+ cells did not adhere and spread within 24 h (Figure 4A). The number of glial cells could not be determined until after 24 h when the cells adhered and showed evidence of spreading into flat aggregates (Figure 4B). Cells at the edge of the clusters tended to extend long processes and expressed classic glial markers, e.g., GFAP, S100b and p75^NTR (F.......

Discussion

EGCs play important roles in gut homeostasis, and it is essential to isolate and study them in vitro. In this protocol, a simple method for isolating EGCs from the lamina propria of the adult mouse intestine was introduced to study enteric glial function.

Removing the adherent mesentery and LMMP with a cotton swab removes some of the inter-myenteric glia residing between the longitudinal and circular muscle, increases the accessibility of buffers to the submucosal surface and removes .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly-D lysine (1 mg/mL stock)	Sigma	A-003-E	Dilute 1:10
Laminin (0.5 mg/mL stock)	Sigma	L4544	Dilute to 10 µg/mL on ICE
EDTA (0.5 M)	Lonza	51201	Dilute 1:100 in DPBS
HEPES (1 M)	Corning	36216004	Dilute 1:100 in DPBS
Cell Recovery Solution	Corning	354253
Dulbecco's Phosphate Buffered Saline (DPBS)	HyClone	SH30028.02
DMEM/F-12	Thermo Fisher Scientific	11320033
Penicillin-Streptomycin (100x)	Life Technologies	15140-122
Gentamicin (50 mg/mL stock)	Life Technologies	15750060
GDNF (10 µg stock)	Sigma	SRP3200
L-Glutamine (200 mM stock)	Life Technologies	25030-081
Chicken anti-GFAP	Thermo Fisher Scientific	PA1-10004
Goat anti-a-Smooth Muscle Actin	Abcam	ab112022
Mouse anti-Pgp9.5	Novus Biologicals	NB600-1160
Goat anti-E-cadherin	R&D Systems	AF748
Rabbit S100	Abcam	ab34686
Mouse p75 NTR	Millipore	MAB5592
Alexa Fluor 488 Goat Anti-Chicken IgY	Invitrogen	A-11039
Alexa Fluor 488 Donkey Anti-Goat IgG	Invitrogen	A-11055
Alexa Fluor 568 Goat Anti-Mouse IgG	Invitrogen	A-11004
Alexa Fluor 594 Donkey Anti-Rabbit IgG	Invitrogen	R-37119
Prolong Gold antifade Reagent with DAPI	Thermo Fisher Scientific	P36931
Fungizone (Amphotericin B) 250 µg/mL	Life Technologies	15290-018
L-Fura-2-AM	Invitrogen	F-14201
CCK peptide	Anaspec, Fremont, CA	AS-20741
Gastrin peptide (Gastrin-17)	Abbiotec, Bloomington, IN	350188
Nylon Mesh Celll Strainer (100 µm)	Fisher Scientific	22363549
Nylon Mesh Celll Strainer (40 µm)	Fisher Scientific	22363547
Disposable Serologic Pipet 5 mL	Fisher Scientific	13-678-11D
0.25% Trypsin-EDTA (1x)	Life Technologies	25200-056