We would like to acknowledge the Broad Stem Cell Research Center FACS and especially thank Jessica Scholes and Felicia Codrea for their help with cell sorting. The work was funded by CIRM RN2-00904-1, K08 HL074229, American Thoracic Society/COPD Foundation ATS-06-065, The Concern Foundation, The UCLA Jonsson Comprehensive Cancer Center Thoracic Oncology Program/Lung Cancer SPORE, the University of California Cancer Research Coordinating Committee and the Gwynne Hazen Cherry Memorial Laboratories (BG).

Outline of Steps
1. Dissection of trachea
2. Cleaning trachea and cutting it
3. Enzyme digestion and processing into single cell suspension
4. Staining for FACS and sorting
5. Processing sorted cells for in vivo and in vitro models
1. Dissection of Trachea
<ol>
	<li>Euthanize the mouse with an intraperitoneal injection of 0.1 mg/0.2 cc of ...

<ol>
	<li>Bartlett, J. A., Fischer, A. J., McCray, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+functions+of+the+airway+epithelium.">Innate immune functions of the airway epithelium.</a> Contrib. Microbiol. 15, 147-163 (2008).</li><li>Finkbeiner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathology+of+tracheobronchial+glands.">Physiology and pathology of tracheobronchial glands.</a> Respir. Physiol. 118, 77-83 (1999).</li><li>Hegab, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Stem/Progenitor+Cell+Population+from+Murine+Tracheal+Submucosal+Gland+Ducts+with+Multipotent+Regenerative+Potential.">A Novel Stem/Progenitor Cell Population from Murine Tracheal Submucosal Gland Ducts with Multipotent Regenerative Potential.</a> Stem Cells. , (2011).</li><li>Jeffery, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+features+of+airway+surface+epithelial+cells+and+glands.">Morphologic features of airway surface epithelial cells and glands.</a> Am. Rev. Respir. Dis. 128, S14-S20 (1983).</li><li>Rock, 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=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 12771-12775 (2009).</li><li>Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trop2+identifies+a+subpopulation+of+murine+and+human+prostate+basal+cells+with+stem+cell+characteristics.">Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20882-20887 (2008).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>McQualter, J. L., Yuen, K., Williams, B., Bertoncello, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+an+epithelial+stem/progenitor+cell+hierarchy+in+the+adult+mouse+lung.">Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 1414-1419 (2010).</li><li>Inayama, Y. <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+and+in+vivo+growth+and+differentiation+of+clones+of+tracheal+basal+cells.">In vitro and in vivo growth and differentiation of clones of tracheal basal cells.</a> Am. J. Pathol. 134, 539-549 (1989).</li><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-L1321 (2002).</li><li>Wu, X., Peters-Hall, J. R., Bose, S., Pena, M. T., Rose, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Bronchial+Epithelial+Cells+Differentiate+to+3D+Glandular+Acini+on+Basement+Membrane+Matrix.">Human Bronchial Epithelial Cells Differentiate to 3D Glandular Acini on Basement Membrane Matrix.</a> Am. J. Respir. Cell Mol. Biol. , (2010).</li><li>Ooi, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+a+putative+tumor-initiating+progenitor+cell+population+predicts+poor+prognosis+in+smokers+with+non-small+cell+lung+cancer.">Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.</a> Cancer Res. 70, 6639-6648 (2010).</li></ol>

This technique to isolate duct and basal cells from the airways is important for improving our understanding of airway repair and regeneration and airway diseases. The techniques described here include a few critical steps. The first is the optimized enzymatic digestion period. The second is the creation of a single cell suspension through serial passaging with progressively higher gage needles to prevent cell shearing but to break up cell clumps. The third is the FACS analysis, and gating of cells with appropriate antib...

<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalog 			number</td>
 </tr>
 <tr>
 <td>Complete 			medium 10: 
 DMEM-F-12 			, 50/50, 1X) </td>
 <td>Mediatech</td>
 <td>15-090-CV </td>
 </tr>
 <tr>
 <td>Hepes (15 mM)</td>
 <td>Invitrogen</td>
 <td>15630</td>
 </tr>
 <tr>
 <td>Sodium 			bicarbonate (3.6mM or 0.03%)</td>
 <td>Invitrogen</td>
 <td>25080</td>
 </tr>
 <tr>
 <td>L-glutamine 			(4 mM)</td>
 <td>Mediatech</td>
 <td>25-005-Cl</td>
 </tr>
 <tr>
 <td>Penicillin 			(100 U/ml)</td>
 <td>Mediatech</td>
 <td>30-001-CI</td>
 </tr>
 <tr>
 <td>Streptomycin 			(100 &mu;g/m)</td>
 <td>Mediatech</td>
 <td>30-001-CI</td>
 </tr>
 <tr>
 <td>Amphotericin 			B (0.25 &mu;g/ ml)</td>
 <td>Lonza</td>
 <td>17-836R</td>
 </tr>
 <tr>
 <td>Insulin 			(10 &mu;g/ml)</td>
 <td>Sigma</td>
 <td>I6634</td>
 </tr>
 <tr>
 <td>Transferrin 			(5 &mu;g/ml)</td>
 <td>Sigma</td>
 <td>T1147</td>
 </tr>
 <tr>
 <td>Cholera toxin 			(0.1 &mu;g/ml)</td>
 <td>Sigma</td>
 <td>C8052</td>
 </tr>
 <tr>
 <td>Epidermal 			Growth Factor (25 ng/ml)</td>
 <td>BD</td>
 <td>354001</td>
 </tr>
 <tr>
 <td>Bovine 			Pituitary Extract (30 &mu;g/ml)</td>
 <td>Invitrogen</td>
 <td>13028-014</td>
 </tr>
 <tr>
 <td>Fetal 			Bovine Serum (5%)</td>
 <td>Fisher</td>
 <td>SH3008803HI</td>
 </tr>
 <tr>
 <td>Retinoic 			acid (0.05 &mu;M)</td>
 <td>Sigma</td>
 <td>R2625</td>
 </tr>
 <tr>
 <td>Growth Factor 			Reduced Matrigel</td>
 <td>BD</td>
 <td>354230</td>
 </tr>
</tbody>
</table>
Table 1. Complete media components.
<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Pronase</td>
 <td>Roche</td>
 <td>10165921001</td>
 <td>Used 			at 0.15%: 
 -o/n 			at 4 &deg;C 			digestion to isolate total tracheal cells (for ALI culture) 
 -4 hr digestion 			4 &deg;C 			to isolate SMG</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>BD 			Biosciences</td>
 <td>354235</td>
 <td>Used 			at 16 Units: 30 min at RT</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Sigma</td>
 <td>DN25</td>
 <td>Used 			at 0.5 mg/ml: 
 20-30 min 			at RT </td>
 </tr>
 </tbody>
</table>
Table 2. Enzymes used for enzymatic digestion of the trachea.

isolation of basal cells and submucosal gland duct cells from mouse trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

isolation-basal-cells-submucosal-gland-duct-cells-from-mouse

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JoVE Journal

Biology

13.3K visualizações.  David Geffen School of Medicine at UCLA. Aqui demonstramos nosso protocolo para isolamento de células basais e das glândulas submucosas duto de traqueias de rato. Nós também demonstram o método de injecção de células estaminais na almofada de gordura dorsal do rato para criar um In vivo Modelo de regeneração da glândula submucosa.