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 Wome...

<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+localization+and+function+of+heme+oxygenase-1+in+cigarette+smoke-induced+cell+death.">Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death.</a> Am. J. Respir. Cell Mol. Biol. 36, 409-417 (2007).</li><li>Kim, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+proteins+regulate+cigarette+smoke+induced+apoptosis:+protective+role+of+heme+oxygenase-1.">Autophagic proteins regulate cigarette smoke induced apoptosis: protective role of heme oxygenase-1.</a> Autophagy. 4, 887-895 (2008).</li><li>Chen, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egr-1+regulates+autophagy+in+cigarette+smoke-induced+chronic+obstructive+pulmonary+disease.">Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.</a> PLoS ONE. 3, e3316-e3316 (2008).</li><li>Okuwa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+micronucleus+assay+for+cigarette+smoke+using+a+whole+smoke+exposure+system:+A+comparison+of+smoking+regimens.">In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens.</a> Exp Toxicol Pathol. , (2009).</li><li>St-Laurent, J., Proulx, L. I., Boulet, L. P., Bissonnette, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+in+vitro+models+of+cigarette+smoke+exposure.">Comparison of two in vitro models of cigarette smoke exposure.</a> Inhal. Toxicol. 21, 1148-1153 (2009).</li><li>Watson, A. M., Benton, A. S., Rose, M. C., Freishtat, R. J. <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+alters+tissue+inhibitor+of+metalloproteinase+1+and+matrix+metalloproteinase+9+levels+in+the+basolateral+secretions+of+human+asthmatic+bronchial+epithelium+in+vitro.">Cigarette smoke alters tissue inhibitor of metalloproteinase 1 and matrix metalloproteinase 9 levels in the basolateral secretions of human asthmatic bronchial epithelium in vitro.</a> J Investig. Med. 58, 725-729 (2010).</li><li>Rennard, S. I. <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+in+research.">Cigarette smoke in research.</a> Am. J. Respir. Cell Mol. Biol. 31, 479-480 (2004).</li><li>Shapiro, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smoke+gets+in+your+cells.">Smoke gets in your cells.</a> Am. J. Respir. Cell Mol. Biol. 31, 481-482 (2004).</li></ol>

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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		<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.

Watch this Scientific Journal Video about Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface at JoVE.com

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 ...

isolation-mouse-respiratory-epithelial-cells-exposure-to-experimental

Research

JoVE Journal

Medicine

33.7K 조회수.  Harvard Medical School. 폐 상피 세포는 생쥐의 호흡기로부터 격리하고 차별 호흡기 상피의 모델로 공기 - 액체 인터페이스에서 교양 수 있습니다. 프로토콜이 환경 독소에 분자 반응을 연구하기 위해, culturing를 분리하고 주류 담배 연기 이러한 세포를 노출에 대해 설명합니다.