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.

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:
<ul>
	<li>Prepare Ham's F12 Media containing antibiotics. To 250 mL Ham's F12 basal media (Cellgro) add 2.50 mL of a 100 X Penicillin/Streptomycin (102 U/mL-102 mg/mL) solution and 250 &mu;L of a 1000 X Fungizone solution. Store at 4&deg;C for up to 4 weeks.</li>
	<li>Prepare a solution of 0.15% Pronase. Add 15 mg Pronase to 10 mL of Ham's F12 Media containing antibiotics. Make fresh and keep on ice until use.</li>
	<li>Prepare a clean work surface and sterile surgical instruments appropriate for small animal surgery, and a lamellar tissue culture hood for cell isolations. All other equipment is considered standard for animal cell culture including humidified CO2 incubators, cold room, tabletop cell centrifuges, inverted microscope, and disposable plastic pipette and culture ware.</li>
	<li>Prepare collagen I solution (50 &mu;g/mL in 0.02 N acetic acid). Pipette 1.0 mL collagen solution into each well of a 12 well transwell plate (Corning). Wrap in parafilm and let stand overnight on a flat surface at room temperature.</li>
</ul>
<ol>
	<li>Using a commercially-available mouse strain (i.e., C57Bl/6, male 6-8 weeks old), euthanize mice using an approved method of euthanasia such as CO2-induced narcosis. Typically 6 mice will yield enough cells (1.5-2.0 x 105 cells/mouse) to seed a 12-well transwell plate.</li>
	<li>Spray the animal carcasses with 70% EtOH solution to sterilize the field.</li>
	<li>With clean surgical scissors and scalpel, remove the skin around the tracheal area, and expose the trachea. Open the abdomen, cut along the sternum, and remove the rib cage. Remove tissue until the end of the trachea is exposed.</li>
	<li>Place tracheas into a 50 mL conical tube containing 30 mL Ham's F12 media containing antibiotics, on ice.</li>
	<li>In a sterile lamellar flow hood, transfer tracheal tissue to a sterile 100 mm Petri dish containing 10 mL Ham's F12 media containing antibiotics.</li>
	<li>Gently dissect connective tissue with sterile forceps and surgical scissors.</li>
	<li>Transfer tracheal tissue to a new 100 mm Petri dish containing 10 mL Ham's F12 media containing antibiotics to rinse. Cut tracheas along vertical axis to expose lumen.</li>
	<li>Transfer tracheas to a 50 mL tube containing 10 mL 0.15% Pronase solution and incubate overnight at 4&deg;C.</li>
	<li>On the second day, have ready:
		<ul>
			<li>Prepare a DNAse I solution. To 18 mL of Ham's F12 Media containing antibiotics add 2 mL of a 10 mg/mL Bovine Serum Albumin (BSA) stock solution, and 10 mg of crude pancreatic DNAse I. Make 1 mL aliquots and store at -20&deg;C (thawed on ice before use).</li>
			<li>Prepare Ham's F12 media containing antibiotics with 20% fetal bovine serum (FBS). To 200 mL Ham's F12 basal media (Invitrogen) add 50 mL heat inactivated FBS, 2.5 mL of a 100 X Penicillin/Streptomycin (102 U/mL-102 mg/mL) solution, and 250 &mu;L of a 1000 X Fungizone solution.</li>
			<li>Prepare MTEC Basic Medium containing antibiotics. To 475.5 mL DMEM/F12 basic media (Cellgro) add 7.5 mL 1 M HEPES solution, 10 mL of 200 mM glutamine solution, 2 mL of a 7.5% NaHCO3 solution, 5 mL of a 100 X penicillin/streptomycin solution, 500 &mu;L of a 1000 X Fungizone solution</li>
			<li>Prepare MTEC medium/10% FBS. To 45 mL of MTEC basic medium containing antibiotics, add 5 mL heat inactivated FBS.</li>
		</ul>
	</li>
	<li>Gently rock the tube (from Step 1.8) 10-12 times, and then let it stand for 30-60 minutes at 4&deg;C.</li>
	<li>Add 10 mL Ham's F12 media containing 20% FBS and antibiotics to the tube and rock 12 times.</li>
	<li>Prepare 3 15 mL conical tubes containing 10 mL Ham's F12 media containing 20% FBS and antibiotics.</li>
	<li>Remove tracheas from Pronase solution, setting aside this solution on ice. Transfer tracheas to first conical tube containing Ham's F12 and invert tube 12 times. Repeat this process two more times.</li>
	<li>Combine Pronase solution with the three supernatants from step 1.13 into one 50 mL tube. Discard remaining tissue.</li>
	<li>Centrifuge at 1400rpm (390 x g; Eppendorf 5810R centrifuge equipped with an A-4-62 rotor) for 10 min at 4&deg;C, and discard supernatant.</li>
	<li>Gently resuspend the pellet in 1 mL DNAse solution (100-200 &mu;L/trachea) and incubate 5 min on ice.</li>
	<li>Centrifuge at 1400rpm (390 x g) for 5 min at 4&deg;C, and discard supernatant.</li>
	<li>Resuspend the cell pellet in 8 mL MTEC medium containing 10% FBS</li>
	<li>Plate cell suspension on Primaria Plates (Falcon). Incubate at 37&deg;C in an atmosphere of 95% air, 5% CO2 for 5 hrs (Note: this is a negative selection step for fibroblasts). </li>
	<li>Collect cell suspension from plates and rinse plates twice with 4 mL MTEC containing 10% FBS. Pool cell suspension and washes together in a 50 mL conical tube. </li>
	<li>Conserve 1 mL for cytospin and cell counting. Spin in a tabletop centrifuge for 5 min at 5,000 rpm (Eppendorf 5415D). Remove 500 &mu;L and resuspend pellet in remaining supernatant. Use 100 &mu;L for cell counting using the Trypan Blue vital staining method. Conserve 4 aliquots of 100 &mu;L for cytospin analysis</li>
	<li>Spin remaining 15 mL cell suspension at 1400rpm (390 x g), at 4&deg;C for 10 min.</li>
</ol>
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&deg;C. As needed, prepare Stock solution B (5 &mu;M) by adding 50 &mu;L Stock A, 500 &mu;L BSA solution (100 mg/ mL) and 49.5 mL Hank's balanced salt solution (HBSS). Store in a foil wrapped tube at -80&deg;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 &mu;L Insulin solution (2 mg/mL insulin in 4 mM HCl ), 250 &mu;L Epidermal growth factor solution (5 &mu;g/mL EGF in HBS containing 1 mg/mL BSA), 200 &mu;L bovine pituitary extract (15 mg/mL in HBS containing 1 mg/mL BSA), 50 &mu;L Transferrin solution (5 mg/mL transferrin in HBS containing 1 mg/mL BSA), 50 &mu;L 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.
<ol>
	<li>Remove collagen solution from transwell plates, let stand under hood for 5 min, and wash with PBS 2 times. </li>
	<li> Following centrifugation, resuspend the cell pellet in an appropriate volume of proliferation media (500 &mu;L) to facilitate plating of 7.5 x 104-1.0 x105 cells per well. Pipette 500 &mu;L cell suspension onto the apical surface of the transwell polycarbonate membrane insert. Add 1.5 mL of proliferation media to the basal compartment of the transwell.</li>
	<li> Incubate the submerged MTEC cultures at 37&deg;C in a humidified incubator containing 95% air, 5% CO2 for 7-10 days. </li>
	<li>Wait three days before changing media the first time. Change media every other day. Monitor cultures by visual inspection and measurement of transepithelial cell resistance using an electrode (EVOM Ohmvoltometer, World Precision Instruments, Sarasota, FL). </li>
	<li>When cells appear confluent and epithelial resistance reaches 1000 &Omega;/cm2, the cells are ready to differentiate at ALI.</li>
	<li>Prepare MTEC basal media containing 2% NuSerum. Add NuSerum to MTEC basic medium to a final concentration of 2% V/V. Add retinoic acid Stock B to a final concentration of 1 X 10-7 M before using. Prepare fresh and use within 2 days.</li>
	<li>Allow cell to differentiate for 10-14 days, by removing apical media and replacing the basal media with 750 &mu;L MTEC basal media containing 2% Nuserum and retinoic acid. Change the basal media and wash the apical side with MTEC containing 2% NuSerum every other day.</li>
</ol>
3. Application of Cigarette Smoke to Cultured Epithelial Cells
<ol>
	<li>Expose the apical side of the transwell cultures to mainstream cigarette smoke using a CS cell exposure system (EMI Instruments, Pittsburgh, PA). See Figure 1 for photograph of the apparatus. See Table of specific reagents and equipment for technical specifications. In brief, the apparatus consists of a peristaltic pump which smokes each cigarette in ~7-8 puffs, drawing in the mainstream smoke into a polypropylene line that is connected to a vented exposure chamber. The exposure chamber is maintained at 37&deg;C by circulating water, and maintained at an atmosphere of 5% CO2 on a gas line. The cells can be exposed to the smoke using Kentucky 3R4F research reference filter cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, KY). Typically cells are exposed to the smoke from 1-2 cigarettes, yielding 100-200 mg/m3 of total particulate matter (TPM). Control cultures are exposed to room air for an equivalent exposure period.</li>
	<li>The TPM is measured according to the manufacturer's protocol supplied with the TE-10c smoking machine (Teague Enterprises). In brief, a filter (Pallflex) is placed in an inline stainless steel holder on a sampling tube leading from the sampling port on the smoking chamber to a constant flow pump (sampling unit). An outflow tube connects the sampling unit to a gas meter (Dry Gas Meter, ONM61,67, AEM). The filter is weighed before and after gas sampling. The total material deposited on the filter in mg is normalized for the total volume of air sampled.</li>
	<li>The cells can then be harvested at any time point (i.e., 0-24 hrs) post exposure for standard cell analytical procedures (i.e., Western immunoblot analysis, mRNA analysis, etc.) or microscopy (i.e., confocal or electron microscopy). The culture media can also be analyzed for cytokines or LDH release for cytotoxicity assays.</li>
</ol>
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 &Omega;/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.
<img src="/files/ftp_upload/2513/2513fig1.jpg" alt="Figure 1" /> 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.
<img src="/files/ftp_upload/2513/2513fig2.jpg" alt="Figure 2" /> 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 &alpha;-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.
<img src="/files/ftp_upload/2513/2513fig3.jpg" alt="Figure 3" /> 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

<ol>
	<li>You, Y., Richer, E. J., Huang, T., Brody, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+mouse+tracheal+epithelial+cells:+selection+of+a+proliferative+population.">Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 283, L1315-L1320 (2002).</li><li>Davidson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+epithelial+cells:+isolation+and+culture.">Murine epithelial cells: isolation and culture.</a> J. Cyst. Fibros. 2, 59-62 (2004).</li><li>Davidson, D. J., Kilanowski, F. M., Randell, S. H., Sheppard, D. N., Dorin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+model+of+differentiated+murine+tracheal+epithelium.">A primary culture model of differentiated murine tracheal epithelium.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 279, L766-L778 (2000).</li><li>Rabe, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.;+Global+Initiative+for+Chronic+Obstructive+Lung+Disease.+Global+strategy+for+the+diagnosis,+management,+and+prevention+of+chronic+obstructive+pulmonary+disease:+GOLD+executive+summary.">et al.; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary.</a> Am. J Respir. Crit. Care Med. 176, 532-555 (2007).</li><li>Macnee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+chronic+obstructive+pulmonary+disease.">Pathogenesis of chronic obstructive pulmonary disease.</a> Clin. Chest Med. 28, 479-513 (2007).</li><li>Tuder, R. M., Yoshida, T., Arap, W., Pasqualini, R., Petrache, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art.+Cellular+and+molecular+mechanisms+of+alveolar+destruction+in+emphysema:+an+evolutionary+perspective.">State of the art. Cellular and molecular mechanisms of alveolar destruction in emphysema: an evolutionary perspective.</a> Proc. Am. Thorac. Soc. 3, 503-510 (2006).</li><li>Yao, H., Rahman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+on+the+role+of+inflammation+in+COPD+and+lung+cancer.">Current concepts on the role of inflammation in COPD and lung cancer.</a> Curr. Opin. Pharmacol. 9, 375-383 (2009).</li><li>van der Toorn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+irreversibly+modifies+glutathione+in+airway+epithelial+cells.">Cigarette smoke irreversibly modifies glutathione in airway epithelial cells.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 293, L1156-L1162 (2007).</li><li>Slebos, D. 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No conflicts of interest declared.

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...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ham&#x2019;s F12 Medium 1X</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>MT-10-080-CM</td>
<td>With L-glutamine</td>
</tr>
<tr>
<td>Pen/strep</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-602E</td>
<td>&nbsp;</td>
<tr>
<td>Pronase</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10165921001</td>
<td>Streptomyces griseus</td>
</tr>
<tr>
<td>Collagen I</td>
<td>&nbsp;</td>
<td>BD biosciences</td>
<td>354236</td>
<td>From rat tail</td>
</tr>
<tr>
<td>Acetic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>338826-25</td>
<td>&nbsp;</td>
<tr>
<td>DNaseI</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>DN25-100MG</td>
<td>From Bovine Pancreas</td>
</tr>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1605-100</td>
<td>Fraction V</td>
</tr>
<tr>
<td>Retinoic Acid</td>
<td>&nbsp;</td>
<td>Retinoic Acid</td>
<td>R265-50MG</td>
<td>&nbsp;</td>
<tr>
<td>Hank&#x2019;s Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14175</td>
<td>Without Ca++ or Mg++</td>
</tr>
<tr>
<td>DMEM-F12</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>MT-15-090-CM</td>
<td>Without L-Glutamine or HEPES</td>
</tr>
<tr>
<td>HEPES, 1M in H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>83264-100ML</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513-100ML</td>
<td>200 mM</td>
</tr>
<tr>
<td>Amphotericin B (Fungizone)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1672346</td>
<td>&nbsp;</td>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>16634-50MG</td>
<td>Bovine Pancreas</td>
</tr>
<tr>
<td>Apo-transferrin (human)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1147-100MG</td>
<td>&nbsp;</td>
<tr>
<td>Cholera toxin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C8052</td>
<td>Vibrio Cholerae</td>
</tr>
<tr>
<td>Epidermal growth factor</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354001</td>
<td>Mouse</td>
</tr>
<tr>
<td>Bovine pituitary Extract</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354123</td>
<td>&nbsp;</td>
<tr>
<td>NuSerum</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>355100</td>
<td>&nbsp;</td>
<tr>
<td>Transwell</td>
<td>&nbsp;</td>
<td>Corning Costar</td>
<td>3401</td>
<td>12 mm, 0.4 mm Pore Polycarbonate</td>
</tr>
<tr>
<td>Primaria 100 mm culture dish</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353803</td>
<td>&nbsp;</td>
<tr>
<td>Pallflex membrane</td>
<td>&nbsp;</td>
<td>Pall Life Sciences</td>
<td>EMFAB TX40H120-WW</td>
<td>&nbsp;</td>
<tr>
<td>Smoking Machine</td>
<td>&nbsp;</td>
<td>EMI Services</td>
<td>ATCSALI-1</td>
<td>see Footnote*</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			*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 (<a href="http://www.tse-systems.com">www.tse-systems.com</a>).		</div>

isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface

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.

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

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.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

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Research

JoVE Journal

Medicine

33.8K Views.  Harvard Medical School. 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. 

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

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.