<p class="jove_content">The authors are very thankful to Dr. Richard Inculet for providing the bronchial tissues. Research Ethics Board approvals were obtained from St. Joseph's Healthcare Hamilton and The University of Western Ontario/London Health Sciences Centre (Dr. David McCormack), Tissue and Archives Committee, Department of Pathology. We also thank Ernie Spitzer (Electron Microscopy, McMaster University) for providing TEMs of our ALI cultures, and Daniela Farkas for providing some of the materials needed for immunostaining. This work was funded by a Block term grant from the Ontario Thoracic Society; Dr. Asma Yaghi was supported by an FSORC scholarship, St. Joseph's Healthcare, Hamilton, Ontario, Canada.</p>

<p class="jove_content">Human Bronchial segments provide an abundant source of primary bronchial epithelial cells. In this article we describe a protocol for growth and expansion of Human bronchial epithelial (HBE) cells from freshly isolated human bronchial segments. This protocol consists of five sections:</p>
<ol>
  <li>Coating and Scratching 100mm Culture Plates</li>
  <li>Preparing Bronchial Tissue Explants</li>
  <li>Bronchial Tissue Transplants</li>
  <li>Passage of HBE Cells</li>
  <li>Growing Ciliated HBE Cells on Transwells</li>...

<ol>
	<li>Phillips, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+Cells+on+Transwell+Inserts+-+Tips+and+Techniques+[Internet].">Growing Cells on Transwell Inserts - Tips and Techniques [Internet].</a> <em style='font-style: italic'>Corning Life Sciences, Technical Resources, Online Training</em>. , (2008).</li><li>Turi, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+activates+anion+exchange+protein+2+and+AP-1+in+airway+epithelial+cells.">Oxidative stress activates anion exchange protein 2 and AP-1 in airway epithelial cells.</a> <em style='font-style: italic'>Am J Physiol Lung Cell Mol. Physiol</em>. <b>283</b>, L791-L798 (2002).</li><li>Lechner, J. F., Haugen, A., McClendon, I. A., Pettis, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+growth+of+normal+adult+human+bronchial+epithelial+cells+in+a+serum-free+medium.">Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium.</a> <em style='font-style: italic'>In Vitro</em>. <b>18</b>, 633-642 (1982).</li><li>Yoshisue, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+ciliated+bronchial+epithelium+1,+a+ciliated+cell-associated+gene+induced+during+mucociliary+differentiation.">Characterization of ciliated bronchial epithelium 1, a ciliated cell-associated gene induced during mucociliary differentiation.</a> <em style='font-style: italic'>Am. J. Respir. Cell Mol. Biol</em>. <b>31</b>, 491-500 (2004).</li><li>Wetering, S. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+secretory+leukocyte+proteinase+inhibitor+(SLPI)+production+by+human+bronchial+epithelial+cells:+increase+of+cell-associated+SLPI+by+neutrophil+elastase.">Regulation of secretory leukocyte proteinase inhibitor (SLPI) production by human bronchial epithelial cells: increase of cell-associated SLPI by neutrophil elastase.</a> <em style='font-style: italic'>J. Investig. Med</em>. <b>48</b>, 359-366 (2000).</li><li>Tristram, D. A., Hicks, W., Hard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+syncytial+virus+and+human+bronchial+epithelium.">Respiratory syncytial virus and human bronchial epithelium.</a> <em style='font-style: italic'>Arch. Otolaryngol. Head Neck Surg</em>. <b>124</b>, 777-783 (1998).</li><li>Mattinger, C., Nyugen, T., Schafer, D., Hormann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+serum-free+culture+conditions+for+primary+human+nasal+epithelial+cells.">Evaluation of serum-free culture conditions for primary human nasal epithelial cells.</a> <em style='font-style: italic'>Int. J. Hyg. Environ. Health</em>. <b>205</b>, 235-238 (2002).</li><li>Freshney, R. I., Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Culture of epithelial cells</em>. , 1-23 (1992).</li><li>Freshney, R. I., Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+bronchial+epithelial+cell+cultures+in+Culture+of+specialized+cells+series.">Normal human bronchial epithelial cell cultures in Culture of specialized cells series.</a> <em style='font-style: italic'>Culture of epithelial cells</em>. , 181-196 (1992).</li></ol>

<p class="jove_content">In this study we presented detailed methods for culture and expansion of primary human bronchial epithelial cells. We demonstrated how explants and transplants of bronchial tissue, cultured in media which promote epithelial cell growth, can provide a continuous source of human airway epithelial cells for studies of non-differentiated (submerged) and differentiated (grown on air-liquid interface) cell models. These cells can be utilized in drug testing systems that more closely resemble the cells in their real physiologic...

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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>Albumin from bovine serum, powder</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4919</td>
<td>Use for coating solution</td>
</tr>
<tr>
<td>COLLAGEN TYPE I 0.1% Solution Sterile-filtered</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C8919</td>
<td>Use for coating solution</td>
</tr>
<tr>
<td>Fibronectin from human plasma</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F2006</td>
<td>Use for coating solution</td>
</tr>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS, sterile)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>28888</td>
<td>Use for rinsing tissue and for coating solution</td>
</tr>
<tr>
<td>DMEM/Ham F-12</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D8437</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F1015</td>
<td>Inactivate, then aliquot and freeze (-20&deg;C); use fresh when needed</td>
</tr>
<tr>
<td>Antibiotic-Antimycotic Stabilized</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5955</td>
<td>Aliquot into 2 ml vials and freeze (-20&deg;C); use fresh when needed</td>
</tr>
<tr>
<td>Albumin solution from bovine serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8412</td>
<td>BSA</td>
</tr>
<tr>
<td>Pituitary Extract Bovine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1476</td>
<td>BPE</td>
</tr>
<tr>
<td>Epidermal growth factor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E9644</td>
<td>EGF</td>
</tr>
<tr>
<td>(&plusmn;)-Epinephrine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E1635</td>
<td>&nbsp;</td>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I6634</td>
<td>&nbsp;</td>
<tr>
<td>Tranferrin Human</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8158</td>
<td>&nbsp;</td>
<tr>
<td>Triiodo-L-thyronine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T6397</td>
<td>&nbsp;</td>
<tr>
<td>Hydorcortisone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H0888</td>
<td>&nbsp;</td>
<tr>
<td>Retinoic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R2625</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9935</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E6758</td>
<td>EDTA</td>
</tr>
<tr>
<td>Phosphate buffered saline</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5368</td>
<td>PBS</td>
</tr>
<tr>
<td>70% ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use for disinfecting and cleaning</td>
</tr>
<tr>
<td>24 well Transwells</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3470</td>
<td>&nbsp;</td>
<tr>
<td>Cell Culture Flasks (T75) CLLBND from Corning</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>05-539-104</td>
<td>These flasks have a Cell Bind coating which promotes cell attachment and growth</td>
</tr>
<tr>
<td>Monoclonal Anti-Cytokeratin, pan-FITC antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F3418</td>
<td>Permeabilization: 0.2% TRITON X-100/PBS; Use 1:250 dilution</td>
</tr>
<tr>
<td>Antibody: E-Cadherin (H108)</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-7870</td>
<td><strong>Do not permeabilize;</strong> Use 1:250 dilution and A21207 (Invitrogen) as secondary antibody</td>
</tr>
<tr>
<td>Alexa Fluor 594 donkey anti-rabbit IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21207</td>
<td>Use 1:400 dilution</td>
</tr>
<tr>
<td>Antibody: Monoclonal mouse Anti- &alpha;-Smooth Muscle Actin-Cy3</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>C6198</td>
<td>Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution</td>
</tr>
<tr>
<td>Antibody: Vimentin (RV202)</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>Sc-32322</td>
<td>Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution and T2402 (Sigma- Aldrich) as secondary antibody</td>
</tr>
<tr>
<td>Rabbit anti-mouse TRITC</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>T2402</td>
<td>Use 1:400 dilution</td>
</tr>
<tr>
<td>Antibody: CD31 or PECAM-1 (M-20)</td>
<td>&nbsp;</td>
<td>Santa-Cruz</td>
<td>Sc-1506</td>
<td><strong>Do not permeabilize;</strong> Use 1:200 dilution and Donkey anti-goat IgG-FITC as secondary antibody</td>
</tr>
<tr>
<td>Donkey anti-goat IgG-FITC</td>
<td>&nbsp;</td>
<td>Santa-Cruz</td>
<td>&nbsp;</td>
<td>Use 1:100 dilution</td>
</tr>
<tr>
<td>Vectashield Mounting medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>Refrigerate in the dark; stains nuclei and retains fluorescence during
prolonged storage</td>
</tr>
<tr>
<td>Vectashield Mounting medium</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>Refrigerate in the dark; retains fluorescence during prolonged storage</td>
</tr>
<tr>
<td>Hoechst Stain solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6024</td>
<td>Stains nuclei; use 1:10 dilution and Vectashield H-1000</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			<p class="jove_content"><strong>Other requirements:</strong> incubator, biological safety cabinet (BSC), centrifuge, 100mm culture plates, sterile tubes (15 ml, 50 ml, and 2 ml), sterile pipette tips, scalpel handle and blades, small sharp scissors, lab coats and gloves. These can be obtained from your preferred suppliers.</p>		</div>
		

primary human bronchial epithelial cells grown from explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm<sup>3</sup> pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO<sub>2</sub> humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Watch this Scientific Journal Video about Primary Human Bronchial Epithelial Cells Grown from Explants at JoVE.com

Primary Human Bronchial Epithelial Cells Grown from Explants

<p class="jove_content">Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.</p>

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

primary-human-bronchial-epithelial-cells-grown-from-explants

Research

JoVE Journal

Biology

23.9K vistas.  McMaster University. A continuación se describe un método detallado para el cultivo de primaria células epiteliales bronquiales humanas a partir de explantes de tejido humano vías respiratorias bronquiales incluyendo el crecimiento diferenciado en una interfase aire-líquido. Este método proporciona una fuente abundante de células primarias para investigar el papel del epitelio de las vías respiratorias en la salud pulmonar y la enfermedad humanas.