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Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse
- Publication Year :
- 2018
- Publisher :
- MyJove Corporation, 2018.
-
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.
- Subjects :
- 0301 basic medicine
General Chemical Engineering
Population
Cell Culture Techniques
General Biochemistry, Genetics and Molecular Biology
Enteric Nervous System
03 medical and health sciences
Mice
Laminin
Submucosa
Glial cell line-derived neurotrophic factor
medicine
Animals
education
Biology
Myenteric plexus
education.field_of_study
Lamina propria
Mucous Membrane
biology
Glial fibrillary acidic protein
General Immunology and Microbiology
Chemistry
General Neuroscience
Cell biology
030104 developmental biology
medicine.anatomical_structure
biology.protein
Enteric nervous system
Neuroglia
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....4145112b88c27dd4a081f90b986feb10