Unterstützt von NIH HD052670.

1. Beschichtung von Platten mit ECM-Proteine <ol><li> Unter sterilen Bedingungen mischen 120 ug von Laminin mit 12 ml kaltem sterile 1X PBS pro Platte. </li><li> Nehmen Bioflex unbehandelten Platten und fügen 2ml der Lösung in jede Vertiefung (Endkonzentration von Laminin ist 2 pg / cm 2). Alternativ könnten andere ECM-Proteine ​​verwendet werden, wie Kollagen-1 [10 ug / cm 2], Fibronektin [5 ug / cm 2], Vitronectin [0,5 ug / cm 2] oder Elastin [10 ug / cm 2]. Das Beschichtungsverfahren ist ähnlich zu dem, was sie für Laminin-Beschichtung beschrieben. Sicherstellen, dass die Kavitäten werden vollständig von der Lösung bedeckt. </li><li> Wickeln der Platten mit Kunststoff und mit über 4 ° C auf einer ebenen Fläche über Nacht auf das Laminin zu adsorbieren können auf den Boden der Vertiefungen. Unter diesen Bedingungen können die Platten für mindestens eine Woche aufbewahrt werden, während noch guter ECM-Funktion. </li><li> Am nächsten Tag, unter sterilen Bedingungen, waschen Sie die Vertiefungen 3 mal mit 1X PBS. </li><li&gt; 1 ml 1% BSA in 1X PBS in jede Vertiefung und inkubieren Sie bei 37 ° C für 1h, um unspezifische Bindungsstellen auf den Membranen zu blockieren. </li><li> Spülen Sie die Platten 3 mal mit 1X PBS, um nicht adsorbierten Proteine ​​zu entfernen. </li><li> Halten der Platten bei 37 ° C in DMEM, bis die Zellen aus Schritt 2,14 isoliert. </li></ol> 2. Isolierung von fetalen Lunge Nager Typ II Epithelzellen Am Tag vor Isolation haben die Bildschirm Tassen mit unterschiedlicher Größe Nylon Maschen autoklavierten und fertig. <ol><li> Erhalten fetalen Lunge aus timed-schwangeren Maus / Ratte auf E17-19 Schwangerschaftswoche. Übertragen Sie das Gewebe in einem 50 ml konischen Röhrchen mit DMEM-Medium und stellen Sie ihn auf Eis. </li><li> Machen Verdauung Puffer in einem 50 ml konischen Röhrchen: (10 ml). Für dieses Volumen, wird Lungengewebe von etwa 20 Föten von Maus / Ratte verwendet. 8,5 ml DMEM 0,5 ml 500 mM HEPES pH 7,4 5 mg Collagenase 1 5 mg Collagenase 1A 1 ml Serum vom Huhn, hitzeinaktiviertem </li><li>Gut mischen, bis alle Komponenten aufgelöst werden und durch ein 0,2-Mikrometer-Spritze Filter innerhalb des sterilen Haube. Platzieren Sie die konische Röhrchen mit dem Verdau-Puffer in einem Wasserbad bei 37 ° C </li><li> Entfernen Sie die DMEM-Medium von dem konischen Röhrchen aus Schritt 2.1 und übertragen das Gewebe in einer sterilen Petrischale. </li><li> Hackfleisch in die Lunge und mit einer sterilen Schere oder Rasierklinge, um Gewebeteile weniger als 1 mm und übertragen das Gewebe in den konischen Röhrchen mit dem vorgewärmten Verdau-Puffer aus Schritt 2.3. </li><li> Verdau des Gewebes durch Auf-und Abpipettieren: 100-mal mit 10 ml Pipette 100-mal mit 5 ml Pipette 100-mal mit Pasteurpipette </li><li> Zentrifuge das Homogenat bei 1.300 UpM für 5 Minuten bei Raumtemperatur. </li><li> Entfernen Sie vorsichtig die Überstände durch Absaugen und erneut zu suspendieren das Pellet mit den Zellen in 15 ml DMEM plus 20% FBS. </li><li> Nehmen Sie die Pokale Bildschirm mit 100 -, 30 - und 15-Mikron-Nylon Maschen und Stelle them auf sterile 150 ml Becher. </li><li> Fügen Sie Zellhomogenat aus Schritt 2.8 und der Reihe nach durch 100 Filter -, 30 - und 15-Mikron-Sieb Tassen. </li><li> Sammeln Sie verklumpt nicht gefilterten Zellen aus dem 30 - und 15-Mikron-Maschen nach mehrmaligem Waschen mit DMEM plus 10% FBS, um die Filtration von nicht-epithelialen Zellen zu erleichtern. </li><li> Entsorgen Sie das Filtrat von dem 15-Mikron Maschenweite, die meist enthält Fibroblasten. </li><li> Reinigen weiterer Typ II-Zellen durch Inkubieren von Zellen aus Schritt 2.11 75 cm 2-Kolben für 30 min (approximatelly10 ml Zellsuspension pro Kolben). </li><li> Die überstehende Flüssigkeit und zentrifugiert bei 1300 UpM für 5 Minuten bei Raumtemperatur die Zellen zu pelletieren. </li><li> Entfernen Sie vorsichtig die Überstände durch Absaugen und erneut zu suspendieren das Pellet mit den Zellen in serumfreiem DMEM. Das Volumen der Resuspension hängt von der Menge an Gewebe verarbeitet. Etwa wir Kennzeichen des Zellen von einem Fötus in jeder Vertiefung (2 ml) erhalten wird, um achiVorabend zwischen 60-70% Konfluenz am folgenden Tag. </li><li> Platte die Zellsuspension auf Bioflex Sechs-Well-Platten mit Laminin oder Fibronectin beschichtet. </li></ol> 3. Isolierung von fetalen Nagetier Lungenfibroblasten <ol><li> Führen Sie die gleichen Schritte für Typ II-Zellen beschrieben Trennung bis zu 2,11. </li><li> Nehmen des Filtrats aus dem 15-Mikrometer-Maschensieb (ohne vorheriges Waschen; ungefähre Volumen 10-15 ml) und die Platte auf 75-cm 2 bei 37 ° C für 30-60 min zu ermöglichen Fibroblasten zu haften. </li><li> Saugen Sie die supernantant und ersetzen mit serumfreiem DMEM, Inkubation über Nacht. </li><li> Am nächsten Tag, Ernte der Zellen mit 0,25% (Gew / Vol) Trypsin in 0,4 mM EDTA und sie auf Platte Bioflex Platten mit Fibronectin beschichtet sind. </li></ol> 4. Experimental-System auf mechanische Reize zu bieten Lungenzellen <ol><li> Seed die Zellen (ca. 50% konfluent) auf Bioflex Kulturplatten in serumfreiem DMEM (2 ml / Vertiefung) und lassen Sie sie anbringen und SPREAnzeige für mindestens 6h vor Experimenten. Im Allgemeinen unserem Labor unterhält Zellen in Kultur für rund 24 Stunden vor der Anwendung mechanischer Beanspruchung. Wenn eine bestimmte Anzahl von Zellen für die Experimente erforderlich sind, nach der Isolierung, sind Typ II-Zellen in serumfreiem DMEM 75-cm 2-Kolben und am nächsten Tag, werden die Zellen trypsiniert, gezählt und ausgesät. </li><li> Am nächsten Tag werden Medien mit frischem serumfreiem DMEM (2 ml / Vertiefung) und Bioflex Platten ersetzt werden in einer Flexcell FX-5000 Stamm Einheit angebracht ist. Monoschichten sollte nicht größer als 80% konfluent vor dem Beginn der Experimente. </li><li> Äquibiaxiale Spannung wird an den Membranen aufgebracht. Das Regime der Belastung hängt von Simulation von mechanischen Kräften in vivo abhängig (siehe Diskussion). </li><li> Zellen, die auf nicht-verstreckte Membranen parallel kultiviert angebaut werden als Kontrollen verwendet. </li><li> Am Ende des Experiments kann Monoschichten verarbeitet, um Veränderungen der Genexpression (zB Surfactant Protein C) zu analysierendurch Echtzeit-PCR, Protein Fülle von Western-Blot, etc. Auch kann Monoschichten für Immunzytochemie Experimente fixiert werden. Für diese Technik, nach der Fixierung sind Silastic Membranen aus den Platten geschnitten und auf Objektträger mit 10-20 l Wasser als Montage-Agent vor Permeabilisierung und Inkubation mit Antikörpern. Der Überstand kann auch verwendet werden, um die Anwesenheit von Wachstumsfaktoren, Cytokine usw. zu untersuchen </li></ol> 5. Repräsentative Ergebnisse Abbildung 1 und Abbildung 2 zeigen repräsentative Phasen-Kontrast-Fotos von E18 fetale Maus-Typ II-Zellen isoliert wurde unter Verwendung der Technik in diesem Manuskript beschrieben. 3 zeigt, dass mechanische Belastung induziert die Differenzierung von fötalem Typ II Epithelzellen mit Surfactant-Protein-C als Marker. <img src="/files/ftp_upload/3543/3543fig1.jpg" /> Abbildung 1. Representative Phasenkontrast-Aufnahme direkt nach der Isolierung, welche die verklumpten Erscheinungsbild der fetalen Typ-II-Zellen aufgenommen. <img src="/files/ftp_upload/3543/3543fig2.jpg" /> Abbildung 2. E18 fetalen Typ II Epithelzellen wurden isoliert, wie hier und vergoldet auf Bioflex Platten mit Laminin beschichtet beschrieben. Foto aufgenommen wurde am folgenden Tag nach dem nicht gestreckten Zellen in Paraformaldehyd fixiert wurden. Die Reinheit der Zellen war entschlossen, sein 90 ± 5% durch mikroskopische Analyse von epithelialen Zellmorphologie und Immunfärbung für SP-C. <img src="/files/ftp_upload/3543/3543fig3.jpg" /> 3. Fetal Typ II Epithelzellen wurden in 5% zyklische Dehnung bei 40 Zyklen / min für 16 h ausgesetzt. A) Northern-Blot von Surfactant-Protein C (SP-C)-mRNA-Expression zeigt, dass der Stamm vom Typ II induziert Zelldifferenzierung unter Verwendung verschiedener Substrate ECM . + / - Zeichen repräsentieren Belichtung oder nicht zu strainieren, jeweils. Daten werden als Mittelwert dargestellt + / - SEM, n = 3; * P &lt;0,05 B) Fluoreszenz-Immunzytochemie Bilder zeigen, SP-C-Protein-Spiegel (grün) in der fetalen Typ II-Zellen ausgesetzt sind oder nicht (Kontrolle) mechanisch beansprucht werden.. Die Zellkerne wurden mit DAPI (blau) gegengefärbt. Bar, 10 um. C) Western-Blot-Ergebnisse aus drei Experimenten zeigen, dass die mechanische Dehnung erhöht SP-C-Protein. N = 3; * P &lt;0,05.

<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>

Keine Interessenskonflikte erklärt.

In diesem Manuskript, beschreiben wir eine Methode zur fetalen Typ-II-Epithelzellen und Fibroblasten zu isolieren und sie auf mechanische Stimulation mit dem Apparat Flexcell Stamm freizulegen. Wir haben diese Technik verwendet, um Differenzierung von Epithelzellen zu bewerten und 1,2-Rezeptor und Signalwege durch Dehnung 3-9 aktiviert zu studieren. Darüber hinaus kann dieses Verfahren auch verwendet werden, um Zell-Reaktionen durch mechanische Verletzung 10,11 induziert werden wird. Da...

<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

Mechanische Kräfte in der Gebärmutter mit sich wiederholenden Bewegungen wie Atmung-und Spannungsgefühl durch Flüssigkeit erzeugt werden, sind entscheidend für die normale Entwicklung der Lunge. Ein wesentlicher Bestandteil der Entwicklung der Lunge ist die Differenzierung der alveolaren Typ II Epithelzellen, die Hauptquelle der pulmonalen Surfactant. Diese Zellen auch in Flüssigkeitshomöostase Teilnahme an der Alveolarlumen, Immunabwehr und zu reparieren. Darüber hinaus können distalen Lungenparenchym Zellen direkt auf übertriebene Dehnung werden während der mechanischen Beatmung ausgesetzt nach der Geburt. Allerdings sind die genauen molekularen und zellulären Mechanismen, durch die Lungenzellen spüren, mechanische Reize, um die Entwicklung der Lunge zu beeinflussen und zu fördern Lungenschädigung nicht vollständig verstanden. Hier bieten wir eine einfache und hoher Reinheit Methode, um Typ-II-Zellen und Fibroblasten von Nagern fetalen Lunge zu isolieren. Dann beschreiben wir ein in vitro System, das Flexcell Dehnungseinheit, auf mechanische Reize, um fötale Zellen liefern, die Simulation mechanischer Kräfte infetalen Lunge Entwicklungs-oder Lungenversagen. Dieses experimentelle System bietet ein ausgezeichnetes Werkzeug, um molekulare und zelluläre Mechanismen der fetalen Lunge ausgesetzten Zellen strecken zu untersuchen. Mit diesem Ansatz hat sich unser Labor mehrere Rezeptoren und Signalproteine, die in der Mechanotransduktion Teilnahme an fetalen Lunge Entwicklung und Lungenschädigung identifiziert.

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

Mechanische Kräfte spielen eine Schlüsselrolle in der Entwicklung und Lunge Lungenversagen. Hier beschreiben wir eine Methode, um Nager fetalen Lunge Typ II Epithelzellen und Fibroblasten zu isolieren und sie auf mechanische Stimulation mit ein Exposé<em&gt; In-vitro-</em&gt; System.

Ein experimentelles System, um Mechanotransduktion in fetalen Lunge Zellen zu untersuchen

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

an-experimental-system-to-study-mechanotransduction-fetal-lung

Research

JoVE Journal

Bioengineering

12.9K Aufrufe.  Alpert Medical School of Brown University. Mechanische Kräfte spielen eine Schlüsselrolle in der Entwicklung und Lunge Lungenversagen. Hier beschreiben wir eine Methode, um Nager fetalen Lunge Typ II Epithelzellen und Fibroblasten zu isolieren und sie auf mechanische Stimulation mit ein Exposé In-vitro- System.

Serie: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon Stretch Growth: The Mechanotransduktion des neuronalen Wachstums

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Forschung

Biotechnik

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

Der Aufbau eines Low-Budget-Laser Axotomie System, um Axon Regeneration in Study C. elegans</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lumineszenz-Resonanzenergietransfer zu Konformationsänderungen in Membranproteinen in Säugerzellen exprimiert Studieren

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electro Studium der Lungenkrebszellen unter Verwendung eines Mehrkanal-Zwei elektrischen Feldes Mikrofluidik-Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Die Erzeugung von ESC-derived Maus Airway epithelialen Zellen unter Verwendung von Dezellularisierte Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

Ein experimentelles Protokoll zur Bewertung der Leistung des neuen Ultraschallsonden basierend auf CMUT Technologie in Anwendung auf die Bildgebung des Gehirns

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA-Nanoröhren als ein vielseitiges Werkzeug, semiflexiblen Polymere zu studieren

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Orbital schütteln Kultur von Säugerzellen in O-förmigen Schiffe, einheitliche Aggregate zu produzieren

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.