1Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Cellular and Molecular Pathology, School of Medicine, University of Pittsburgh
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Lam, H. C., Choi, A. M., Ryter, S. W. Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface . J. Vis. Exp. (48), e2513, doi:10.3791/2513 (2011).
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.
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:
2. Propagation and Differentiation of MTEC At Air-Liquid Interface
Prepare retinoic acid stock solutions. Stock solution A: Weigh Retinoic acid in the dark (Mr 300.44 g/mol) to make a 5 mM stock solution in 95% EtOH. Store in a foil wrapped tube at -80°C. As needed, prepare Stock solution B (5 μM) by adding 50 μL Stock A, 500 μL BSA solution (100 mg/ mL) and 49.5 mL Hank's balanced salt solution (HBSS). Store in a foil wrapped tube at -80°C for up to 4 weeks.
Prepare MTEC proliferation medium containing retinoic acid. To 45.7 mL MTEC basal media containing antibiotics, add 2.5 mL heat-inactivated FBS, 1 mL Retinoic acid Stock B, 250 μL Insulin solution (2 mg/mL insulin in 4 mM HCl ), 250 μL Epidermal growth factor solution (5 μg/mL EGF in HBS containing 1 mg/mL BSA), 200 μL bovine pituitary extract (15 mg/mL in HBS containing 1 mg/mL BSA), 50 μL Transferrin solution (5 mg/mL transferrin in HBS containing 1 mg/mL BSA), 50 μL cholera toxin solution (100 mg/mL in HBS containing 1 mg/mL BSA). Filter sterilize final media preparation and use within 2 days of preparation.
3. Application of Cigarette Smoke to Cultured Epithelial Cells
4. Representative Results
Successful epithelial isolation, proliferation, and differentiation at ALI should yield an intact monolayer with a cobblestone morphology. Transepithelilal cell resistance of healthy cultures should be approximately 1000-2000 Ω/cm2. Figure 2 depicts a monolayer of mouse respiratory epithelial cells stained for epithelial cell and cilia markers under control conditions and after cigarette smoke exposure. Figure 3 shows representative electron micrographs of healthy MTEC cultures, depicting ultrastructure and cilia morphology.
Figure 1. Preparation of epithelial cells proceeds through three phases, an isolation step, a propagation stage, and a differentiation stage at air-liquid interface (scheme). Epithelial cells are isolated from mouse trachea, seeded onto transwells where they proliferate in submerged culture, and then converted into air-liquid interface where they fully differentiate. Experiments are typically conducted on the 24th day of continuous culture. The picture depicts a model system for exposure of cultured cells to mainstream cigarette smoke. A transwell ALI culture system is placed inside a custom modular cigarette smoke delivery system which is controlled for temperature, humidity and CO2.
Figure 2. Epithelial cell cultures were fixed and stained for nuclei (Hoechst 33258), F-actin (green; Alexa-Fluor 488-conjugated Phalloidin) and the cilia marker acetylated α-tubulin (red; Cy3-conjugated secondary antibody). The lower panels show equivalent images of epithelial cells taken 4 hours after exposure to cigarette smoke (2 cigarettes, 150 mg/m3). (B) Lactate dehydrogenase (LDH) release was measured in the basal medium 24 hours after exposure to varying doses of cigarette smoke as indicated.
Figure 3. Air-liquid interface cultures of epithelial cells isolated from mouse tracheal epithelium differentiate in several subtypes that by transmission electron microscopy (TEM) have the characteristics of ciliated, basal, and non-ciliated cells 1. These cells typify pseudostratified respiratory epithelium. Figure labels correspond to: bb: basal body, a: axoneme, m: mitochondria, n: nucleus, s: substrate (polycarbonate membrane), mv: microvili
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 described in Step 1.19. Provided that the cultures are monitored, washed, and the culture media replenished every other day following the initial 72 hour incubation, the cultures should fully differentiate in approximately two weeks from the initiation of ALI.
Chronic obstructive pulmonary disease (COPD), which is largely caused by chronic cigarette smoke exposure, continues to represent a major global health problem 4-7. COPD, is characterized by progressive airflow limitation, destructive alveolar loss (emphysema), and exaggerated inflammatory responses of the lung to cigarette smoke 4-7. A large number of studies have used alveolar, bronchial, and airway epithelial cell systems to attempt to model lung cell responses to CS. Many of these studies have been conducted in epithelial cell or transformed lines (i.e., Beas-2b), see Refs 8-11 for examples. The ALI transwell culture system allows the culture of pulmonary epithelial cells in fashion that is closer to their physiological orientation in the airway, than that which can be provided by conventional liquid (submerged) culture 1-3. Although the featured protocol describes the application of this system to mouse tracheal primary cultures 1, in principle other primary epithelial cells can be applied (i.e., mouse Type II epithelial cells, Human bronchial epithelial cells, etc.). The application of live mainstream CS to cell cultures represents a model that perhaps more closely approximates human exposure to CS than application of aqueous cigarette smoke extract (CSE), which is commonly used in cellular studies of CS exposure 12-15. CSE represents a soluble fraction of mainstream smoke that lacks many chemical components of whole smoke. While both CS exposure systems have limitations, CSE has the advantage of being more easily calibrated, since a single preparation can be used for multiple experiments, and dosing is achieved by dilution 15-16. On the other hand, we include here a method for quantifying mainstream smoke delivery based on TPM measurements. The elucidation of molecular and cellular responses to toxin exposure, in particular CS as exemplified in this article, will further the understanding of the impact of air pollution on human health, in particular the etiology of chronic diseases of the lung.
No conflicts of interest declared.
We thank Emeka Ifedigbo for technical assistance and Dr. Shivraj Tyagi for valuable expertise. We also thank the Harvard NeuroDiscovery Center for assistance with microscopy. This work was supported in part by an American Heart Association Predoctoral grant 09PRE2250120 to Hilaire Lam, and NIH grants, R01-HL60234, R01-HL55330, R01-HL079904, awarded to A. M. K. Choi.
|Ham’s F12 Medium 1X||Cellgro||MT-10-080-CM||With L-glutamine|
|Pronase||Roche Group||10165921001||Streptomyces griseus|
|Collagen I||BD Biosciences||354236||From rat tail|
|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|
|Amphotericin B (Fungizone)||Fisher Scientific||1672346|
|Cholera toxin||Sigma-Aldrich||C8052||Vibrio Cholerae|
|Epidermal growth factor||BD Biosciences||354001||Mouse|
|Bovine pituitary Extract||BD Biosciences||354123|
|Transwell||Corning||3401||12 mm, 0.4 mm Pore
|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).