Supported by NIH grant HD052670.

1. Coating of plates with ECM proteins
<ol>
	<li>Under sterile conditions, mix 120 &mu;g of laminin with 12 ml of cold sterile 1X PBS per plate.</li>
	<li>Take Bioflex untreated plates and add 2ml of the solution to each well (final concentration of laminin is 2 &mu;g/cm2). Alternatively, other ECM proteins could be used, such as collagen-1 [10 &mu;g/cm2], fibronectin [5 &mu;g/cm2], vitronectin [0.5 &mu;g/cm2] or elastin [10 &mu;g/cm2]. The coating technique is sim...

<ol>
	<li>Sanchez-Esteban, J., Tsai, S. W., Sang, J., Qin, J., Torday, J. S., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+forces+on+lung-specific+gene+expression.">Effects of mechanical forces on lung-specific gene expression.</a> Am. J. Med. Sci. 316, 200-204 (1998).</li><li>Sanchez-Esteban, J., Cicchiello, L. A., Wang, Y., Tsai, S. W., Williams, L. K., Torday, J. S., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+promotes+alveolar+epithelial+type+II+cell+differentiation.">Mechanical stretch promotes alveolar epithelial type II cell differentiation.</a> J. Appl. Physiol. 91, 589-595 (2001).</li><li>Sanchez-Esteban, J., Wang, Y., Cicchiello, L. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+stretch+inhibits+cell+proliferation+and+induces+apoptosis+in+fetal+rat+lung+fibroblasts.">Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 282, L448-L456 (2002).</li><li>Sanchez-Esteban, J., Wang, Y., Cicchiello, L. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+stretch+inhibits+cell+proliferation+and+induces+apoptosis+in+fetal+rat+lung+fibroblasts.">Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 282, L448-L456 (2002).</li><li>Sanchez-Esteban, J., Wang, Y., Gruppuso, P. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+induces+fetal+type+II+cell+differentiation+via+an+epidermal+growth+factor+receptor-extracellular-regulated+protein+kinase+signaling+pathway.">Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway.</a> Am. J. Respir. Cell. Mol. Biol. 30, 76-83 (2004).</li><li>Wang, Y., Maciejewski, B. S., Lee, N., Silbert, O., McKnight, N. L., Frangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+fetal+type+II+epithelial+cell+differentiation+is+mediated+via+cAMP-PKA-dependent+signaling+pathway.">Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 291, L820-L827 (2006).</li><li>Sanchez-Esteban, J., Wang, Y., Filardo, E. J., Rubin, L. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+{beta}1,+{alpha}6,+and+{alpha}3+contribute+to+mechanical+strain-induced+differentiation+of+fetal+lung+type+II+epithelial+cells+via+distinct+mechanisms.">Integrins {beta}1, {alpha}6, and {alpha}3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 290, L343-L350 (2006).</li><li>Wang, Y., Maciejewski, B. S., Soto-Reyes, D., Lee, H. S., Warburton, D., Sanchez-Esteban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+promotes+fetal+type+II+epithelial+cell+differentiation+via+shedding+of+HB-EGF+and+TGF-alpha.">Mechanical stretch promotes fetal type II epithelial cell differentiation via shedding of HB-EGF and TGF-alpha.</a> J. Physiol. 587, 1739-1753 (2009).</li><li>Wang, Y., Maciejewski, B. S., Drouillard, D., Santos, M., Hokenson, M. A., Hawwa, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+caveolin-1+in+mechanotransduction+of+fetal+type+II+epithelial+cells.">A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 298, L775-L783 (2010).</li><li>Lee, H. S., Wang, Y., Maciejewski, B. S., Esho, K., Fulton, C., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-10+protects+cultured+fetal+rat+type+II+epithelial+cells+from+injury+induced+by+mechanical+stretch.">Interleukin-10 protects cultured fetal rat type II epithelial cells from injury induced by mechanical stretch.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 294, L225-L232 (2008).</li><li>Hawwa, R. L., Hokenson, M. A., Wang, Y., Huang, Z., Sharma, S., Sanchez-Esteban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+inhibits+inflammatory+cytokines+released+by+fetal+mouse+lung+fibroblasts+exposed+to+mechanical+stretch.">IL-10 inhibits inflammatory cytokines released by fetal mouse lung fibroblasts exposed to mechanical stretch.</a> Pediatric Pulmonology. , (2011).</li><li>Kitterman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+forces+on+fetal+lung+growth.">The effects of mechanical forces on fetal lung growth.</a> Clin. Perinatol. 23, 727-740 (1996).</li><li>Harding, R., Crystal, R. G., West, J. B., Banes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+breathing+movements.">Fetal breathing movements.</a> The Lung: Scientific Fountations. , 2093-2104 (1997).</li><li>Harding, R., Liggins, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+thoracic+dimensions+induced+by+breathing+movements+in+fetal+sheep.">Changes in thoracic dimensions induced by breathing movements in fetal sheep.</a> Reprod. Fertil. Dev. 8, 117-124 (1996).</li></ol>

In this manuscript, we describe a method to isolate fetal type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using the Flexcell Strain Apparatus. We have used this technique to assess differentiation of epithelial cells 1,2 and to study receptor and signaling pathways activated by stretch 3-9. In addition, this method can also be used to investigate cell responses induced by mechanical injury 10,11. The procedure is based on digestion of the lung tissue ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1A</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screen cups</td>
			<td>Sigma</td>
			<td>CD1-1KT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-754-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100 micron nylon mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-100-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 micron mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-30-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 micron mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagen-1</td>
			<td>Collagen Biomed</td>
			<td>PC0701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Sigma</td>
			<td>V-0132</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elastin</td>
			<td>Sigma</td>
			<td>E-6402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioflex plate</td>
			<td>Flexcell International</td>
			<td>BF-3001U</td>
			<td>Uncoated</td>
		</tr>
		<tr>
			<td>Flexcell Strain Unit</td>
			<td>Flexcell International</td>
			<td>FX-5000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an experimental system to study mechanotransduction in fetal lung cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Watch this Scientific Journal Video about An Experimental System to Study Mechanotransduction in Fetal Lung Cells at JoVE.com

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

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Bioengineering

12.9K Views.  Alpert Medical School of Brown University. Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.