Diese Forschung wird von der CounterACT Programm, National Institutes of Health (NIH), das Büro des Direktors, und dem National Institute of Environmental Health Sciences (NIEHS) Grant Number U54 ES015678 (CWW) unterstützt. SA wird auch von Kinderkrankenhaus Colorado / Colorado School of Mines Collaboration Pilot-Award # G0100394 und Kinderklinik Colorado Forschung Institue Pilot-Award # G0100471 unterstützt.

1. Rat Cardiomyocyte Kulturen <ol><li> Alle Experimente wurden unter Protokolle, die von der institutionellen Pflege der Tiere und die Verwendung Ausschuss genehmigt IACUC durchgeführt. </li><li> Erhalten Rattenherzmuskelzellen aus den Herzen (Ventrikel) der männlichen Ratten (240-260 g) unter Verwendung zuvor 4 beschriebenen Verfahren. Kurz gesagt, Tiere anästhesiert unter Verwendung einer intraperitonealen Injektion von Pentobarbital (100 mg / kg; bestätigen Anästhesie durch Zehenklemmverfahren) und dann Herzen in 10,0 ml, 1 mM Ca 2 + enthält, Krebs-Ringer-Puffer, pH 7,4, zu entfernen. </li><li> Spülen Sie die Herzen 5-6x, um Blut zu entfernen und dann wechseln Sie zu einem Ca 2 +-freien Krebs-Ringer-Puffer mit 0,02% und 0,06% Protease Kollagenase A (5,0 ml / Herz). Die Herzen in dieser Lösung für 10-15 min bei 37 ° C unter gelegentlichem Schütteln. </li><li> Nach 10-15 min, waschen die enzymatische Lösung mit Ca 2 +-freien Krebs-Ringer-Puffer für weitere 5 min. Lassen Sie die Zellen aus dem Gewebe schlaff unsten eine 25-ml-Pipette durch Pipettieren der Suspension nach oben und unten mehrmals. </li><li> Trennen Sie die Zellen von Gewebe durch Filtrieren durch eine 70 um Nylon-Mesh und es ihnen ermöglichen, in Krebs-Ringer-Puffer mit 0,1 mM Ca 2 + zu begleichen. Aussetzung des Zellpellets in 1,0 ml Krebs-Ringer-Puffer, enthaltend 0,2 mM Ca 2 +. </li><li> Vorsichtig Schicht über 5,0 ml 60 ug / ml Rinderserumalbumin, BSA, in 15 ml Zentrifugenröhrchen, zu Kardiomyozyten aus nonmyocytes trennen und es ihnen ermöglichen, für 30 Minuten absetzen. Die Herzmuskelzellen sind schwerer und bewegen sich auf den Boden des Röhrchens. Entfernen Sie vorsichtig den Überstand Zellen und Medien. Wiederholen Sie diesen Schritt einmal, um weiter zu reinigen, die Zellen. </li><li> Schrittweise Übergang durch Waschen (in den Schritten unter Verwendung von 0,25 mM, 0,5 mM, 0,75 mM und 1,0 mM Ca 2 +-haltigen Puffer) die ventrikulären Myocyten / Kardiomyozyten bis 1,0 mM Ca 2 +-haltigen Puffer und ACCT Medium, bestehend aus Dulbecco modifiziertem Eagle-Medium resuspendiert , DMEM containing 2 mg / ml BSA, 2 mM L-Carnitin, 5 mM Kreatin, 5 mM Taurin, 100 IU / ml Penicillin und 100 ug / ml Streptomycin. </li><li> Platte der Kardiomyozyten in ACCT Medium in einer Dichte von 100 bis 150 Zellen / mm 2 auf 100 mm oder 35 mm Laminin beschichteten Kunststoffkulturschalen oder 40 x 22 mm Deckgläschen mit Laminin (1 ug / cm 2) schwemmt. Nach 1 Stunde, waschen das Geschirr mit 2,0 ml ACCT zu Zellen, die nicht gebunden sind, zu entfernen. In 10,0 ml frisches Medium und Inkubation der Zellen. </li></ol> 2. Differenzierte Air-Liquid-Interface (ALI) Kultur der Menschen Airway Epithelial Cells Basal <ol><li> Procure menschlichen Geweben tracheobronchiale von National Disease Research Interchange unter Institutional Review Board genehmigt Protokolle. Zellernte und durchzuführen, Kultur mit einem veröffentlichten Verfahren 5,6. Dieses Verfahren verwendet Zellkulturen, wie sie sind ein präzises Modell für den menschlichen Einatmung von TIC jedoch, wenn nötig kann man isolieren und zu wachsen ALI mozu verwenden und / oder Ratte Epithelzellen der Atemwege 7,8. </li><li> Kurz gesagt, stellen kleine Stücke (~ ¼ Inch) des Gewebes nach dem Entfernen aller Bindegewebe und Lymphknoten. Mehrmals in Ringer-Laktat-Lösung waschen Gewebe. Hinzufügen Gewebe zu einer 50 ml konischen Röhrchen und fügen Protease-Lösung (1% Protease/0.01% DNase in Minimalmedium, MEM, Gewebe Fluidverhältnis (1:10)). </li><li> Rock the Gewebe bei 4 ° C über Nacht. Enden die Dissoziation des Gewebes durch Zugabe von 10% fötalem Rinderserum. Kratzen die Epitheloberfläche mit einem chirurgischen Skalpell und Sammeln der Zellen durch Zentrifugation (2.000 Upm für 10 min). </li><li> Platten Zellen bei Passage auf einem kollagenbeschichteten snapwells in speziellen ALI Medium hergestellt, wie ausführlich von Fulcher et al 9 beschrieben. Kultur für 5 Tage vor dem Wechsel zum Luft-Flüssigkeit-Grenzfläche (ALI) durch Entfernen apikalen Medium. </li><li> Füttern Sie die Zellen mit ALI Medien jeden zweiten Tag und damit die Zellen für weitere 2-3 Wochen (zu unterscheidenbevor Expositionen beobachten zahlreiche Schläge Cilien und Schleim Sekretion). Waschen Sie die apikale Oberfläche mit warmem PBS zusammen mit Medienwechsel. </li></ol> 3. Chlor Belichtungs <ol><li> Enthält die Chlor-Belichtungssystem (CES) in einem qualifizierten chemischen Abzugshaube mit einer Betriebsgeschwindigkeit von 100 Gesicht fpm, die die notwendige Auffang Exposition von Personal im Falle des zufälligen Chlor Lecks zu verhindern bietet. </li><li> Betreiben Sie das System unter leichten Überdruck (0,5 Zoll Wasser). Trockene Luft bei 15 L / min und einem angemessenen Niveau von Chlor zugeführt wird, um die gewünschte endgültige Chlorkonzentration zu erreichen zugeführt. Die Kammern haben einen Verschlussdeckel mit 4 Mini-BCU Schleusen und einem niedrigen Härtegrad Silikondichtung, um den Druck Dichtung zu schaffen. </li><li> Geben Sie sich die Cl 2-Gemisch durch einen Massendurchflussregler. Die CES verwendet eine Druckgasflasche, die 1,0% Cl 2 in trockenem Stickstoff. </li><li> Regulieren Sie den Luftstrom mit Hilfe der Verdünnungkundenspezifische Systemsteuerung und ähnlich regeln die Cl 2-Konzentration in den Expositionskammern geliefert. Ein geringes Volumen Probenahmepumpe zieht die Abgase aus den Kammern in eine Chlor-Analysator zu Konzentrationen, die dann auf einem Datenlogger an den Analysator angeschlossen aufgezeichnet werden, zu überwachen. </li><li> Messen Sie die Durchflussmengen in den Kammern vor der Belichtung mit einem Durchflussmesser gleich und Abgasraten zu gewährleisten. </li><li> Bereiten Sie die Zellkulturen für die Exposition durch Entfernen der überstehenden Medien und Zugabe von frischem Medium (basolateralen Medien in ALI-Kulturen). Alle Vorbehandlungen mit Agenten konnte zu diesem Zeitpunkt durchgeführt werden. </li><li> Expose die ALI Kulturen der Epithelzellen der Atemwege zu Cl 2-Gas (50, 100 oder 300 ppm für 30 Minuten) in den beiden verschlossenen Polysulfon biocontainment Kammern. Die Herzmuskelzellen (untergetaucht oder konfluenten Kulturen auf Membranen) auf 50 oder 100 ppm Cl 2 für 15 Minuten ausgesetzt. </li><li> Nach der Belichtung bündig die Kammern mit Luft, bis das Cl 2 Stand unter 1 ppm und sicher geöffnet werden, um Zellen (innerhalb von 5 min) zu entfernen. </li></ol> 4. Transepitheliale Elektrischer Widerstand (TER) Mess <ol><li> Messen Sie die TER der Luft-Flüssigkeit-Grenzfläche, ALI, Kulturen mit einem epithelialen voltohmmeter mit einem Paar von Silberchlorid &quot;Stäbchen&quot;-Elektroden. </li><li> Äquilibrieren Sie die Essstäbchen Elektrode in der ALI Medien 15 min vor dem Gebrauch. </li><li> In warmen Medien zur apikalen (1,0 ml) und basolateralen (2,0 ml) und messen Sie die Oberfläche mit den Ess-Stäbchen TER Elektroden der voltohmmeter. </li><li> Tauchen Sie den kürzeren Arm der Elektrode in apikalen Medien und den längeren Arm in den basolateralen Medien. Klicken Sie auf die &quot;Maßnahme&quot;-Taste auf der voltohmmeter, den elektrischen Widerstand zu beurteilen. </li><li> Die voltohmmeter hat die Möglichkeit, Ohm oder k Ohm messen. Subtrahieren den Widerstand über einem zellfreien Kulturträger aus dem Widerstand über jede Zelle gemessen Schicht trans zu ergebenepithelialen Widerstand (TER). </li></ol> 5. Caspase Mess <ol><li> Add frischen ALI Medien (2,0 ml) zur basolateralen Oberfläche nach der Belichtung und Inkubation der Zelle bei Raumtemperatur. Sammeln Stand Medien bei 4 und 24 Stunden. </li><li> Messen Sie Caspase 3/7 Aktivität in den Medienüberständen mit Hilfe eines kommerziellen Caspase 3/7 Assay-Kit. </li></ol> 6. Western Blot und Immunzytochemie <ol><li> Führen Western Blots mit Zelllysaten wie zuvor beschrieben 10. Hängen Sie das Proteinlysat (20 ug) in 5x Probenpuffer reduziert und kochen für 5 min. Betreff des Proteinlysat SDS-PAGE (4-15%) und die Übertragung der getrennten Proteine ​​auf eine Nitrocellulosemembran durch elektrophoretische Blot. </li><li> Blockieren nicht-spezifischer Bindung durch Inkubation der Membran mit 5% Milch in Waschpuffer (PBS + 0,1% Detergens) und die Sonde die Membranen mit primärem Antikörper gegen Actin SERCA2 oder sarcomeric bei 1:1000 Verdünnung, über Nacht bei 4 ° C. Weiter, waschen und Inkubation mit den jeweiligen Membranen Peroxidase-konjugierten Sekundärantikörper zu entwickeln und für den Nachweis nach wie vor mit handelsüblichen 10-Peroxidase-Nachweis-Kit beschrieben. </li><li> Für Immunzytochemie Behandlung lebenden Zellen auf Einlagen oder Deckgläschen in 6-Well-Platten für 20 min nach dem Spülen mit PBS mit 0,4% gewachsen Triton-X-100 in 10 mM Natriumcitrat-Puffer. </li><li> Block unspezifische Bindung durch Behandlung der Zellen mit 5% Eselserum für 20 min inkubiert und dann die Zellen mit nicht-spezifischen IgG oder einzelne primäre Antikörper spezifisch sarcomeric Actin oder Ki-67. </li><li> Waschen der Zellen mit PBS und Inkubation mit Fluoreszenz-konjugierte sekundäre Antikörper an den primären Antikörper und einem Nuklearfarbstoff (1 &amp;mgr; g / ml DAPI) zu erfassen. </li><li> Mit PBS waschen und montieren Deckgläser mit handelsüblichen Montage Medien. </li><li> Visualisieren Sie die Färbung mit einem Fluoreszenzmikroskop. </li></ol>

<ol>
	<li>Mohan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+accidental+exposure+to+chlorine+gas:+clinical+presentation,+pulmonary+functions+and+outcomes.">Acute accidental exposure to chlorine gas: clinical presentation, pulmonary functions and outcomes.</a> Indian J Chest Dis Allied Sci. 52, 149-152 (2010).</li><li>Kose, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction,+acute+ischemic+stroke,+and+hyperglycemia+triggered+by+acute+chlorine+gas+inhalation.">Myocardial infarction, acute ischemic stroke, and hyperglycemia triggered by acute chlorine gas inhalation.</a> Am J Emerg Med. 27, 1021-1024 (2009).</li><li>Das, R., Blanc, P. 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The authors declare that they have no competing financial interests.

Die häufigste Form der akuten toxischen Expositionen tritt auf, wenn man eine giftige Chemikalie in die Lunge atmet. Diese Chemikalien können auch schnell in den Blutkreislauf aufgenommen werden und andere Organe wie Herz und Gehirn beeinflussen. Giftigkeit beim Einatmen von verschiedenen Agenten mit Hilfe von Tiermodellen untersucht und darüber berichtet, aber die Mechanismen sind weniger gut verstanden. Dies ist eine große Hürde bei der Entwicklung wirksamer Therapien. Das Fehlen von in-vitro-Belichtungssyste...

Inhalativen Exposition gegenüber toxischen Chemikalien (TIC) / Gase wie Chlor (Cl 2) bleibt eine laufende Gesundheitsproblem in zufälligen Positionen als auch in ihren möglichen Einsatz als chemische Bedrohung Mittel. Obwohl die Lunge das primäre Ziel sind Organe wie Herz und Gehirn betroffen 1-3. In vivo-Modelle sind in der Regel für die Prüfung Toxizität von TIC verwendet, aber in vitro-Assays zur Beurteilung Toxizität sind einfacher, schneller und kostengünstiger. In vitro-Modellen auch für die umfangreiche Untersuchung von Mittel-Zell-Interaktionen, die schwer zu in vivo zu beurteilen sein kann zu ermöglichen. Wie in vitro-Belichtungssysteme sind selten und außerdem in einigen herkömmlichen Modellen, bei denen giftige Substanzen werden dem Kulturmedium der Zellen hinzugefügt taucht, die Eigenschaften der Mittel kann aufgrund von Wechselwirkungen und die Bindung an Komponenten in dem Medium ändern. In solchen Szenarien Zellkultursystemen, wie Luft-Flüssigkeit-Grenzfläche (ALI) Kulturen von primären humanen Epithelzellen der Atemwege, hier vorgeschlagen, die direkt in den gasförmigen Mittel ausgesetzt werden könnte vielversprechend. Epithelzellen der Atemwege sind die ersten Verteidigungslinien gegen giftige Chemikalien eingeatmet. Der menschliche Luftwegsepithel bildet eine physikalische Barriere zwischen dem Lumen und den darunterliegenden Zellen in der Lunge und nimmt an der Reaktion der Lunge. Es produziert eine Anzahl von Cytokinen und anderen pro-und anti-inflammatorische Mittel sowie Schleim sekretiert / Atemwegsoberflächenflüssigkeit (ASL), die das Epithel. Eine der Einschränkungen bei herkömmlichen in vitro-Kultursystemen untergetaucht ist auch, dass die ASL und Schleim, die Epitheloberfläche Abdeckung entfernt oder verdünnt. Dies entspricht nicht den physiologischen Zustand der Lungen Epithelzellen, die der Luft ausgesetzt sind. Daher sollte ein ideales in vitro-System für die TIC Toxizität dieser Architektur repliziert werden. Es gibt ein großes Interesse an der Entwicklung schnelle Screening methoden, die in vivo-Toxizität vorherzusagen. Epithelzellen an der ALI gewachsen zu differenzieren und haben gut differenzierten Strukturen und Funktionen im Vergleich zu Zellen gewachsen untergetaucht und servieren ein überlegenes Modell der Atemwege. In dieser Studie beschreiben wir den Einsatz von Luft-Flüssigkeit-Grenzfläche Kultur der Menschenatemwege (tracheobronchiale) Epithelzellen zum Testen eingeatmeten Giftgastoxizität und vergleichen Sie sie mit einem untergetauchten Zellkultur von Kardiomyozyten, damit das Studium ein weiteres wichtiges Ziel der Toxizität.

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

Zellkulturen sind unverzichtbar für die Entwicklung und Studie die Wirksamkeit von therapeutischen Mitteln, vor ihrem Einsatz in Tiermodellen. Wir haben die einzigartige Fähigkeit, gut differenzierten humanen Atemwegsepithel und Herzmuskelzellen zu modellieren. Dies könnte ein wertvolles Werkzeug, um die schädlichen Wirkungen toxischer inhalierten Chemikalien wie Chlor, die normalerweise mit der Zelloberfläche interagieren, und bilden verschiedene Nebenprodukte bei der Reaktion mit Wasser, und ihren Effekten in Submerskulturen zu studieren. Unser Modell mit gut differenzierten menschlichen Atemwege epithelialen Zellkulturen bei Luft-liqiuid Schnittstelle umgeht diese Beschränkung auf und bietet die Möglichkeit, kritische Mechanismen der Toxizität von möglichen giftigen Chemikalien inhalativen beurteilen. Wir beschreiben eine verbesserte Verlust der Membranintegrität, Caspase Mitteilung und Tod auf toxische chemische inhalativen Exposition wie Chlor. In diesem Artikel schlagen wir Methoden, um Chlor Exposition in Säugetier-Herz und Atemwegsepithelzellen c modellierenEllen in Kultur und einfache Tests, um ihre Wirkung auf diese Zelltypen zu evaluieren.

Primäre stabförmigen Kardiomyozyten befestigen auf Laminin Matrizen und zu verbreiten und differenzieren sich in konfluenten Kulturen (Abbildung 1A und seinen Kasten). Diese Zellen wurden weiter auf der Grundlage des sarko Aktin und SERCA2 Ausdruck (Fig. 1B und 1C) gekennzeichnet. Kardiomyozyten sind sehr anfällig für Chlor Toxizität als 15 min Einwirkung von 100 ppm Chlor verursachte umfangreiche Zellabrundung und Tod in Submerskulturen und Störungen der konfluen...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Dieses Protokoll wurde entwickelt, um Belichtungsverfahren von Zellkulturen zu inhalierenden toxischen Chemikalien zeigen. Exposition der differenzierten Luft-Flüssigkeit-Schnittstelle (ALI) Kulturen von Epithelzellen der Luftwege bietet eine einzigartige Modell von Atemwegs Exposition gegenüber toxischen Gasen, wie Chlor. In dieser Handschrift beschreiben wir Wirkung von Chlor-Exposition auf die Luft-Flüssigkeits-Grenzkulturen von Epithelzellen und Tauchkultur der Herzmuskelzellen. In-vitro-Belichtungssysteme erlauben wichtige mechanistische Studien, um Wege, die dann genutzt werden, um neue therapeutische Mittel zu entwickeln beurteilen.

In-vitro-Zellkulturmodell für Toxic Inhalative Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

in-vitro-cell-culture-model-for-toxic-inhaled-chemical-testing

Research

JoVE Journal

Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K Aufrufe.  University of Colorado.  Dieses Protokoll wurde entwickelt, um Belichtungsverfahren von Zellkulturen zu inhalierenden toxischen Chemikalien zeigen. Exposition der differenzierten Luft-Flüssigkeit-Schnittstelle (ALI) Kulturen von Epithelzellen der Luftwege bietet eine einzigartige Modell von Atemwegs Exposition gegenüber toxischen Gasen, wie Chlor. In dieser Handschrift beschreiben wir Wirkung von Chlor-Exposition auf die Luft-Flüssigkeits-Grenzkulturen von Epithelzellen und Tauchkultur der He...

Serie: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Mikrofluidik-Vorrichtung für die Neuerstellung eines Tumor-Mikroumgebung<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Forschung

Biotechnik

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Eine Methode zur Ovarialfollikel Encapsulation und Kultur in einem Proteolytisch Degradable 3 Dimensional-System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Formgedächtnis-Polymere für Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autologe endotheliale Vorläuferzellen Cell-Sätechnik und Biokompatibilitätstestung Für Herz-Kreislauf-Geräte in Großtiermodell

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotierende Cell Culture Systems für Human Cell Culture: Human Trophoblastzellen als Modell

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Test auf Augenreizung (EIT) zur Kennzeichnung der Gefahr von Augen Reizt Chemicals mit rekonstruierter menschlicher Hornhaut-ähnlichen Epithelial (RhCE) Gewebemodell

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Licht-vermittelte Bildung und Musterung von Hydrogelen für Zellkulturanwendungen

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Ein<em&gt; In Vitro</em&gt; Organ Culture Modell der Murine Bandscheiben

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Methodik für biomimetische Chemie Neuromodulation der Ratte Netzhaut mit dem Neurotransmitter Glutamat In-vitro-

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Ein menschliches 3D extrazelluläres Matrix-Adipozyten-Kulturmodell zum Studium der Matrix-Zell Metabolischen Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...