Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Summary

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Abstract

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Protocol

The overall protocol requires 2 days for cell isolations from animal tissue, 5-10 days for cell proliferation, and an additional 10-14 days for cell differentiation at air-liquid interface. An additional day is required for cell exposures and harvesting of samples.

1. Isolation of Mouse Tracheobronchial Epithelial Cells (MTEC).

Note: All procedures described below have been reviewed and approved by the Institutional Animal Care and Use Committee at Brigham and Women's Hospital/ Harvard Medical School Area.

Before getting started:

• Prepare Ham's F12 Media containing antibi....

Discussion

The protocol describing isolation of mouse tracheal epithelial cells is adapted from the protocols of You et al., ¹, and others ^2-3 with modifications. As with any protocol describing cell isolations, the most critical aspect is to avoid contamination from bacterial or fungal pathogens by using strict aseptic techniques. A second critical step is to avoid fibroblast contamination of the cultures, which can be avoided by careful dissection of the tracheas, and negative selection as describe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ham’s F12 Medium 1X	Cellgro	MT-10-080-CM	With L-glutamine
Pen/strep	Lonza Inc.	17-602E
Pronase	Roche Group	10165921001	Streptomyces griseus
Collagen I	BD Biosciences	354236	From rat tail
Acetic Acid	Sigma-Aldrich	338826-25
DNaseI	Sigma-Aldrich	DN25-100MG	From Bovine Pancreas
Bovine Serum Albumin	Fisher Scientific	BP1605-100	Fraction V
Retinoic Acid	Retinoic Acid	R265-50MG
Hank’s Balanced Salt Solution	GIBCO, by Life Technologies	14175	Without Ca++ or Mg++
DMEM-F12	Cellgro	MT-15-090-CM	Without L-Glutamine or HEPES
HEPES, 1M in H2O	Sigma-Aldrich	83264-100ML
L-Glutamine	Sigma-Aldrich	G7513-100ML	200 mM
Amphotericin B (Fungizone)	Fisher Scientific	1672346
Insulin	Sigma-Aldrich	16634-50MG	Bovine Pancreas
Apo-transferrin (human)	Sigma-Aldrich	T1147-100MG
Cholera toxin	Sigma-Aldrich	C8052	Vibrio Cholerae
Epidermal growth factor	BD Biosciences	354001	Mouse
Bovine pituitary Extract	BD Biosciences	354123
NuSerum	BD Biosciences	355100
Transwell	Corning	3401	12 mm, 0.4 mm Pore Polycarbonate
Primaria 100 mm culture dish	Falcon BD	353803
Pallflex membrane	Pall Corporation	EMFAB TX40H120-WW
Smoking Machine	EMI Services	ATCSALI-1	see Footnote*
*The cigarette smoking machine is a custom designed and fabricated 14"x14"x20" Dual chambered and water jacketed light tint clear proof 1/2" thick polycarbonate Lexan chamber for cigarette smoke exposure with temperature controlled, water level sensor controlled shut off system. A cigarette smoking/puffing unit is installed for a variable cigarette puffing rates. When the unit is in use, it mimics an incubator in the sense that the temperature, humidity and carbon dioxide are controlled in the system. The system includes: (I) a customized dual chamber/water jacketed unit that maintains a controlled environment for tissue culture experiments. (II) A digital heavy duty, high precision dual pump water temperature circulator system with water level sensor and temperature control (III) A cigarette smoking unit with puffing pump. (IV) A pump cycle sensor control rate cycler (IV) A Stainless steel high precision in-Line filter holder. (V) A Detachable lid mounted 11/2" size axial uniformity cigarette smoke mixing fan. (VI) A medium size water bath with mounting bracket for the water circulator. (VII) A 1/2’ thick Plexiglas tray with brackets for the puff pump and holder for cigarette ash collector. This machine as described can be substituted with similar commercially-available smoking machines such as the kind available from TSE systems (www.tse-systems.com).