Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Summary

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Abstract

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in ¹. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases ². The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands ³. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways ³.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Protocol

Outline of Steps

1. Dissection of trachea

2. Cleaning trachea and cutting it

3. Enzyme digestion and processing into single cell suspension

4. Staining for FACS and sorting

5. Processing sorted cells for in vivo and in vitro models

1. Dissection of Trachea

1. Euthanize the mouse with an intraperitoneal injection of 0.1 mg/0.2 cc of pentobarbital.
2. Cut open the abdominal wall, and move the intestines to the side to expose the abdominal aorta and then dissect it.

Discussion

This technique to isolate duct and basal cells from the airways is important for improving our understanding of airway repair and regeneration and airway diseases. The techniques described here include a few critical steps. The first is the optimized enzymatic digestion period. The second is the creation of a single cell suspension through serial passaging with progressively higher gage needles to prevent cell shearing but to break up cell clumps. The third is the FACS analysis, and gating of cells with appropriate antib.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Complete medium 10: DMEM-F-12 , 50/50, 1X)	Mediatech	15-090-CV
Hepes (15 mM)	Invitrogen	15630
Sodium bicarbonate (3.6mM or 0.03%)	Invitrogen	25080
L-glutamine (4 mM)	Mediatech	25-005-Cl
Penicillin (100 U/ml)	Mediatech	30-001-CI
Streptomycin (100 μg/m)	Mediatech	30-001-CI
Insulin (10 μg/ml)	Sigma	I6634
Transferrin (5 μg/ml)	Sigma	T1147
Cholera toxin (0.1 μg/ml)	Sigma	C8052
Epidermal Growth Factor (25 ng/ml)	BD	354001
Bovine Pituitary Extract (30 μg/ml)	Invitrogen	13028-014
Fetal Bovine Serum (5%)	Fisher	SH3008803HI
Retinoic acid (0.05 μM)	Sigma	R2625
Growth Factor Reduced Matrigel	BD	354230
Table 1. Complete media components.
Pronase	Roche	10165921001	Used at 0.15%: -o/n at 4 °C digestion to isolate total tracheal cells (for ALI culture) -4 hr digestion 4 °C to isolate SMG
Dispase	BD Biosciences	354235	Used at 16 Units: 30 min at RT
DNase I	Sigma	DN25	Used at 0.5 mg/ml: 20-30 min at RT
Table 2. Enzymes used for enzymatic digestion of the trachea.