Name: improving 2d and 3d skin in vitro models using macromolecular crowding
Uploaded: 2016-08-22T13:00:00
Description: Watch this Scientific Journal Video about Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding at JoVE.com

This work was supported by the Biomedical Research Council, Singapore through core support to the Institute of Medical Biology and grant SPF2013/005. M.R. was supported by a NUS Faculty Research Committee Grant (Engineering in Medicine) (M.R.) R-397-000-081-112, and the NUS Tissue Engineering Program (NUSTEP). The authors would like to thank Professor Irene Leigh for providing the collagen VII antibody. Electron microscopy work was carried out at the National University of Singapore Electron Microscopy Unit.

1. Makromolekulare Crowding in 2D Hautzellkulturen <ol><li> Seed 50.000 Zellen (primäre Fibroblasten oder primäre Keratinozyten oder Co-Kultur von primären Fibroblasten und Keratinozyten) pro Well einer 24-Well-Platte. Samenzellen in 1 ml der entsprechenden Zelltyp Wachstumsmedien. </li><li> Erlauben Zellen über Nacht anhaften, in einem 37 ° C Inkubator bei 5% CO 2. </li><li> Entsorgen Sie alte Medien und ersetzen mit 1 ml frischem Medium molekularen Crowders und 100 uM Ascorbinsäure enthält. Verwend...

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Enhanced extracellular matrix is obtained upon introduction of macromolecular crowders to cell culture, owing to the excluded volume effect which increases the propeptide cleavage by proteinases. This results in extracellular matrix, in particular collagen, to be processed faster and deposited on the culture surface. While other groups have obtained thick fibroblast cell layers, it involved a culture time of several weeks20. In contrast, MMC described in this Protocol, dramatically shortens the culture time wh...

Die Haut bildet eine schützende Barriere, die durch Wasserverlust und Krankheitserreger Eintritt zu verhindern. Es besteht aus drei Hauptkomponenten; die Stroma reichen Dermis 1, eine geschichtete Epidermis 2,3,4 oben drauf, und der dermo-epidermalen Verbindung zwischen 5,6. Die Dermis besteht hauptsächlich aus kollagenen und elastischen Fasern zusammengesetzt und ist dünn besiedelt mit Fibroblasten 7. Im Gegensatz dazu wird die zellreiche Epidermis aus mehreren Schichten von Keratinozyten zusammen. Die Keratinozyten der innerste Schicht sind proliferative und neue basalen Zellen bereitzustellen, die unheilbar erneuern und ersetzen Keratinozyten, die sich ständig bewegen, um die äußerste Schicht der Haut und haben verloren ihre Kerne und Zytoplasma-Material, was zu einer verhornte Schicht Differenzierung, die erfährt Schuppung. Die dermale-epidermale Verbindung, eine bestimmte Art von Basalmembran, ist eine komplexe Struktur aus miteinander verbundenen Matrixmolekülen zusammengesetzt, die tethers die Epidermis in die Dermis. Collagen-I-Fasern der Dermis Interlace mit Kollagen VII Fibrillen Verankerung, die an das Kollagen IV reichen Lamina densa verankert sind. Verankerungs Filamente (Laminin 5, Kollagen XVII und Integrine) wiederum verbinden die Lamina densa mit Hemidesmosome basaler Keratinozyten. Basalen Keratinozyten (Stratum basale) haben die Fähigkeit, sich zu vermehren und zu erneuern, sowie differentiate und stratify die suprabasalen Schichten zu bilden; stratum spinosum, stratum granulosum, und schließlich das Stratum corneum, die mit der Umgebung der Kontaktfläche der Haut dar. Auf dem Weg von der Basalschicht an die Hornschicht der Schalter Keratinozyten Expressionsmuster von Zytokeratine und schließlich Apoptose und umhüllen sich in verhornten Umschläge Rümpfe der spezifischen Proteine, die durch Transglutaminase-Aktivität kovalent vernetzt sind. Recreating Haut und deren Schichten in vitro, einschließlich der komplexen Strukturen des<html> dermalen-epidermalen Verbindung und dermalen extrazellulären Matrix und die Verhornungsprozess zu emulieren, hat lange faszinierte Wissenschaftler und bioengineers als anspruchsvolle Aufgabe. Es wurden bedeutende Fortschritte in Hautgewebe - Engineering, beispielsweise die erfolgreiche Extraktion von Hautzellen von Patienten Biopsien und die Erzeugung von Haut organotypischen Kulturen unter Verwendung von Patienten stammenden Hautzellen 8. Jedoch bleiben ungelöste Probleme in Bezug auf schlechte Sekretion von extrazellulären Matrixproteinen von den Hautzellen selbst und was zu suboptimalen Hautmodelle. Weiterhin wird die Zeit variiert zwischen vier bis acht Wochen 3D organotypischen Haut Kokultur mit den aktuellen Protokolle zu erzeugen, einen Zeitrahmen, die potentiell mit dem Einbau von hochmolekularen Crowders verkürzt werden konnte. Reduktionskultivierungszeit spart Reagenzkosten, verringert die Häufigkeit von Zellalterung und reduziert die Wartezeit des Patienten sollte in der Klinik das Produkt verwendet werden. t &quot;&gt; makromolekulare Verdrängung (MMC) mit spezifischen Makromolekülen in das Kulturmedium einzuführen ausgeschlossen Volumeneffekte zu erzeugen. Diese beeinflussen die enzymatische Reaktionsraten einschließlich der proteolytischen Spaltung von Prokollagen , die 9-13 säumig unter wässrigen Standardkulturbedingungen ist. Unter MMC, enzymatische Reaktionen 14,15 was im Falle von Prokollagen Spaltung, eine erhöhte Menge an Kollagen I Moleküle in gedrängten Kulturen im Vergleich zu Kontrollen laufenen 10. da die Umwandlung von Prokollagen zu Kollagen beschleunigt , ohne die Menge der Reagenzien Erhöhung ermöglicht die Bildung von Kollagen Baugruppen, mit MMC kultivierten Fibroblasten 48 Stunden ergab signifikant mehr Kollagen I im Vergleich zu laufenen Fibroblastenkulturen für bis zu vier Wochen überwacht 11,16,17. Neben Auswirkungen auf enzymatische Aktivitäten, die die Bildung, Stabilisierung und Remodellierung von ECM, MMC auch beeinflussen wurde Kollagen gezeigt, direkt verbessern und zu modulierenFaserbildung 18,19. Wir stellen Ihnen hier eine Methode, um die extrazelluläre Matrix (ECM) Produktion von Hautzellen zu verbessern, insbesondere dermale Fibroblasten und Keratinozyten. Darüber hinaus zeigen wir, dass die angereicherte ECM unter MMC in Monolayer-Kulturen erzeugt werden, können als reine Zellen abgeleiteten Matrix (CDM) dezellularisiert und verwendet werden. Wir verwenden eine nicht-konventionellen Ansatz zu visualisieren und in vollem Umfang schätzen die von Hautzellen kultiviert abgelagert ECM mit MMC. Interferenzreflexionsmikroskopie zur Untersuchung von Zell-Matrix-Interaktionen oder Zell-zu-Glas-Kontaktstellen der Regel verwendet. Diese Technik wurde in unserem System verwendet, um die Gesamtmenge der Matrix, auf der Glasoberfläche abgeschieden anzuzeigen. Interferenz-Reflexionsmikroskopie wurde gekoppelt mit Fluoreszenzimmunfärbungs die meisten Informationsmenge in Bezug auf die extrazelluläre Matrixzusammensetzung und das Muster, in Gegenwart und Abwesenheit von MMC zu erhalten. Organotypic Haut Kokulturen ist eine klassische Methode , um die Haut in vitro in einem dreidimensionalen Kontext zu modellieren. Während zweidimensionale Kokulturen signifikante Information liefern kann, ist sie begrenzt , wenn diese Daten zu übersetzen und es zurück zu einer in vivo - Umgebung der Anwendung, die von Natur aus eine dreidimensionale Struktur. Haut-Keratinozyten, insbesondere sind polarisiert und enthalten apikalen und basalen Segmente, die für die Homöostase und Zelladhärenz wesentlich sind. Zusätzlich ist die Expression von typischen suprabasalen Proteine ​​in Keratinozyten über der basalen Schicht, wie Keratin 1, Keratin 10 und Filaggrin nur im Zuge der Schichtung und terminale Differenzierung von Keratinozyten. Da die terminale Differenzierung kaum in typischen Monolayer-Kulturen ist, wird suprabasal Proteinexpression normalerweise nicht in diesem Kultursystem erreicht. Daher beginnen organotypischen Kulturen in Kulturmedium eingetaucht, aber werden dann einer Luft-Flüssigkeit-Schnittstelle angehoben zu fahren keratinocyte Differenzierung. Dies resultiert in der Expression von Markern Schichtung sogar Verhornung und eine allgemein bessere Reflexion der epidermalen Physiologie. Während andere Gruppen zuvor organotypischen Haut Co-Kulturen erfolgreich generiert haben, hat sich die Einrichtung eines funktionalen dermoepidermale Verbindungszone ein Thema. Hier präsentieren wir eine neue Methode zur Kultivierung von organotypischen Haut Co-Kulturen mit einer verbesserten Basalmembran, in einer verkürzten Zeitrahmen und ohne die Reife dieser Konstrukte zu beeinträchtigen. Dies würde Haut Mimetika für in - vitro - Modellierung bereitzustellen, die Untersuchung der Hautbiologie und ein Sortiment von Screening - Assays.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ascorbic acid</td>
			<td style="width:181px;">Wako</td>
			<td style="width:175px;">013-12061</td>
			<td style="width:499px;">Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture inserts&#160; (6-well format)</td>
			<td style="width:181px;">Greiner Bio-One</td>
			<td style="width:175px;">657610</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrate solution</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">S2369</td>
			<td style="width:499px;">DakoCytomation Target Retrieval Solution Citrate, pH 6 (x10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagen (rat tail)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">354236</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">CnT-57&#160;</td>
			<td style="width:181px;">CELLnTEC</td>
			<td style="width:175px;">CnT-57</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DAB Substrate + chromogen kit</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">K3468</td>
			<td>Histology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Deep-well plate (6-well format)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">355467</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">ECL detection reagent</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">RPN2106</td>
			<td>Amersham ECL Western Blotting Detection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Electron microscope (TEM)</td>
			<td>&#160;JEOL&#160;</td>
			<td>&#160;JEM-1010 (100kV)</td>
			<td>Transmission electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fibroblast media (FM)</td>
			<td></td>
			<td></td>
			<td>High Glucose-DMEM + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM70</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0310-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM400</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0300-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Keratinocyte serum-free media (KSFM)</td>
			<td style="width:181px;">Life Technologies</td>
			<td style="width:175px;">17005-042</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Lysis buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">89900</td>
			<td style="width:499px;">Lysis buffer for protein extraction. A protease inhibitor (Roche, #11836170001) was added to this lysis buffer.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;">LSM510</td>
			<td style="width:499px;">Red, green and blue channels to visualize AF594, AF488 and DAPI fluorescent immunostainings (40X mag).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Hydromount)</td>
			<td style="width:181px;">National Diagnostics</td>
			<td style="width:175px;">HS-106</td>
			<td>Fluorescent staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Cytoseal)</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">8310-16</td>
			<td>Histology (HRP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">OCT compound (Tissue Tek)</td>
			<td style="width:181px;">Sakura</td>
			<td style="width:175px;">4583</td>
			<td>Embedding for cryotomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-streptomycin antibiotics</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">A5955</td>
			<td>Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Primary antibodies</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen I antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab6308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen Type IV</td>
			<td style="width:181px;">Novocastra</td>
			<td style="width:175px;">#NCL-COLL-IV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-collagen VII</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>LH7.2 (in house)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Fibronectin antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab2413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Reducing agent&#160;</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0009</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sample buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Secondary antibodies (Immunostaining)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">#D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11037&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">&#160;#A-11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 chicken-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Secondary antibodies (HRP)</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">Envision + System - HRP Labelled Polymer Anti-Rabbit and Anti-Mouse</td>
			<td>undiluted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sodium deoxycholate</td>
			<td style="width:181px;">Prodotti Chimicie Alimentari</td>
			<td style="width:175px;"></td>
			<td>Decellularization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Stratification media</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ham&#8217;s F12&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10% fetal bovine serum&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">100U/mL penicillin and 100U/mL streptomycin&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">0.4mg/mL hydrocortisone&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL insulin</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1.8&#183;10-4 M adenine&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL transferrin&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">2&#183;10- 11 M triiodothyronine&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Trypsin</td>
			<td style="width:181px;">Biopolis Shared Facilities</td>
			<td style="width:175px;">0.125% Trypsin/Versene&#160;&#160; pH 7.0 + 0.3&#160;</td>
			<td>Used to trypsinize cells</td>
		</tr>
	</tbody>
</table>

improving 2d and 3d skin in vitro models using macromolecular crowding

Das Glycoprotein-Familie von Kollagenen stellt die Hauptstrukturproteine ​​in den menschlichen Körper, und sind wichtige Komponenten von Biomaterialien in modernen Tissue Engineering eingesetzt. Ein technischer Engpass ist die Ablagerung von Kollagen in vitro, da sie bekanntermaßen langsam ist, in suboptimalen Bildung von Bindegewebe und anschließende Gewebekohäsions resultierende, insbesondere in Hautmodellen. Hier beschreiben wir ein Verfahren, das die Zugabe von differentiell-sized Saccharose Co-Polymere Hautkulturen umfasst makromolekulare Verdrängung (MMC) zu erzeugen, die in einer dramatischen Erhöhung der Kollagenablagerung führt. Insbesondere abgeschieden dermalen Fibroblasten eine erhebliche Menge an Kollagen I / IV / VII und Fibronektin unter MMC im Vergleich zu Kontrollen. Das Protokoll beschreibt auch ein Verfahren gedrängten Zellschichten zu Dezellularisieren, beträchtliche Mengen an extrazellulären Matrix (ECM) Belichten der auf der Kulturfläche zurückgehalten wurden, wie durch immunocy bewiesentochemistry. Gesamtmatrixmasse und Verteilungsmuster wurde unter Verwendung von Interferenz-Reflexions-Mikroskopie untersucht. Interessanter, Fibroblasten, Keratinozyten und Co-Kulturen hergestellt Zellen abgeleiteten Matrizen (CDM) der Zusammensetzung und der Morphologie verändern. CDM könnte als &quot;bio-Scaffolds&quot; für die sekundäre Zellaussaat, wo die aktuelle Verwendung von Beschichtungen oder Gerüste, typischerweise von xenogenen tierischen Quellen verwendet werden, vermieden werden können, damit zur weitere klinisch relevante Anwendungen bewegen. Zusätzlich beschreibt dieses Protokoll die Anwendung von MMC während der Tauchphase eines 3D-organotypischen Haut Kokultur-Modell, das ausreichend war ECM Ablagerung in der dermo-epidermalen Verbindung (DEJ), insbesondere, Kollagen VII, der Hauptbestandteil zu verbessern von Ankerfibrillen. Die Elektronenmikroskopie bestätigte die Anwesenheit von mit MMC entwickelten Fibrillen in Kulturen Verankerung, im Vergleich zu Kontrollen. Dies ist bedeutsam, als Verankerungs Fibrillen der Dermis auf die Epidermis anbinden, damitmit einem vorgeformten reifen DEJ Hauttransplantation Empfänger in Bezug auf die Graft-Stabilität und die gesamte Wundheilung profitieren können. Des Weiteren wurde Kulturzeit von 5 Wochen bis 3 Wochen kondensiert ein reifes Konstrukt zu erhalten, wenn MMC verwenden, die Kosten zu senken.

Makromolekulare crowding konnte ECM Ablagerung zu verbessern, insbesondere Fibroblasten mehr Kollagen I abgeschieden, IV und Fibronectin im Vergleich zu Kulturen Kontrolle (Abbildung 1, Cell Schicht; 1A, Kollagen I; 1B, Kollagen IV, 1C, Fibronektin). Nach Dezellularisierung, war es offensichtlich , dass Fibroblasten die Haupt Einleger von Kollagen I waren, IV und Fibronectin im Vergleich zu Keratinozyten (Abbildu...

Watch this Scientific Journal Video about Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding at JoVE.com

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Wir stellen eine Protokoll-Zellen abgeleiteten Matrices reich an extrazellulären Matrixproteinen zu erhalten, unter Verwendung von hochmolekularen Crowders (MMC). Darüber hinaus präsentieren wir ein Protokoll, das MMC in 3D organotypischen Haut Co-Kultur-Generation enthält, die Kulturzeit reduziert, während Laufzeit von Konstrukt beibehalten wird.

Die Verbesserung der 2D- und 3D-Haut<em&gt; In Vitro</em&gt; Modellen mithilfe von Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

The overall goal of this procedure is to enhance extracellular matrix deposition and improve on current 2D and 3D skin models using macromolecular crowding. Crowding improves the generation of cell-derived matrices, and mature organotypic cultures in a condensed time frame. This method can answer key questions in the field of tissue engineering because it offers improved ways of generating cell-derived extracellular matrices for the bioengineering of skin and other tissues.The main advantage of this technique is that cell-derived matrices and organotypic skin constructs can be generated in a significantly condensed time frame when applying macromolecular crowding to these cultures. The technique of interference reflection microscopy allows you to visualize the total extracellular matrix deposition without the need of antibody staining. Applications of these techniques will extend from studies of extracellular matrix deposition by skin cells to improved wound therapies, especially with the generation of better skin equivalents and tissue culture for grafting.To begin, seed 50, 000 primary fibroblasts, primary keratinocytes, or a co-culture of both, in one milliliter of medium pro well, into the wells of a 24-well plate. Allow the cells to adhere in a 37 degree Celsius, 5%carbon dioxide incubator overnight. Then, discard the old medium and replace it with one milliliter of fresh medium containing macromolecular crowders and 100-micromolar ascorbic acid.Incubate the cultures for six days, changing the medium every two days. Carry out immunofluorescence and Western blotting according to the text protocol. To make and use a cell-derived matrix, after culturing the cells for six days with molecular crowders and ascorbic acid as just demonstrated, decellularize the cell layers to obtain a cell-derived matrix by first using 1X PBS to wash the cell layers twice.Aspirate the PBS, and then add 250 microliter of 0.5%sodium deoxycholate and incubate on ice for 10 minutes. Aspirate the cell lysate, then add an additional 250 microliter of 0.5%sodium deoxycholate and repeat the 10-minute incubation and aspiration two more times. Use distilled water to wash the cell-derived matrix twice.Aspirate the water, and then add 1X PBS. Store the matrix at four degrees Celsius for up to one week. To prepare a secondary cell suspension, aspirate the PBS from the matrix, and seed 50, 000 keratinocytes on top of the F mat for example.Allow the cells to adhere overnight. To carry out interference reflection microscopy, or RIM, on a cell-derived matrix, perform image acquisition using a con-focal microscope according to the text protocol. To cast a collagen gel with fibroblasts encapsulated in a cell culture insert, aliquot 10 milliliters of rat tail collagen type one into a cold beaker.Add one milliliter of cold DMEM and 0.5 milliliters of one molar sodium hydroxide to neutralize the acidic collagen. Then add 0.5 milliliters of fibroblast suspension. Using a cold pipette, evenly divide the 12 milliliters of solution into cell culture inserts in a six-well culture plate.Incubate the gel at 37 degrees Celsius and 5%carbon dioxide for one hour. Once the gel has solidified, submerge it in fibroblast medium or FM.Then after incubating it for 24 hours, aspirate the FM.Use four milliliters of fresh medium containing macromolecular crowders to replace the old FM by adding two milliliters to the interior of the cell culture insert and two milliliters to the outside of the cell culture insert. Following a 24-hour incubation, use 0.125%trypsin cheating agent to trysonize the keratinocytes.Tap the sides of the flask gently to dislodge the cells and add serum-containing medium to neutralize the trypsin. After counting the cells, prepare a keratinocyte suspension. Next, aspirate the medium from the cell culture inserts, and add the keratinocytes to the top of the gel.Incubate the gel for one hour. After the keratinocytes have attached to the gel surface, use two milliliters of KSFM, or CTN-57 serum-free medium to submerge the cell inside the gel culture insert and add two milliliters of FM to the outside of the cell culture insert. Following a 24-hour incubation, discard the old medium and replace it with fresh medium containing macromolecular crowders.After seven days in the submerged culture, to raise the culture to an air-liquid interface, transfer the culture insert to a deep-well plate. Add 10 milliliters of stratification medium to the outside of the cell culture insert, keeping the interior of the insert dry. Finally, incubate the cultures and change the medium every three days for 14 days.Harvest and process them according to the text protocol. As shown here, macromolecular crowding allowed fibroblasts to deposit more extracellular matrix or ECM as compared to control cultures. Upon secularization, it was evident that fibroblasts were the main depositors of collagen one, collagen four, and fibronectin.This figure demonstrates that is was fibroblasts rather than keratinocytes that were the main depositors of collagen seven, and this is the first report of successful collagen seven deposition in vitro. Using RIM, the full extent of the cell-derived matrix was captured. An overlay of the RIM image with antibody staining shows the relative quantity of that ECM component in relation to the total amount of ECM.In a 3D in vitro skin model, macromolecular crowding or MMC, shortened the culture time from five weeks to three, to obtain a mature organotypic skin co-culture. As seen here, by H and E staining, at three weeks, the culture with MMC consisted of a pleuristratified epidermis and a stromol ridge dermis. In addition, intense and continuous collagen seven immnunostaining was detected at the dermalepidermal junction of crowded cell cultures as compared to a weak and spotty collagen seven immunostaining in control cultures.Transmission electron microscopy also show the presence of anchoring fibrils in organotypic cultures generated with MMC, showing functional collagen seven. Cell-derived matrices could be used for secondary cell seeding purposes as an alternative scaffold as the complexity of the extracellular matrix is maintained, and it is produced by cells themselves. Organotypic skin cultures, which are generated under macromolecular crowding, could be used as an improved 3D skin model as it contain a stromal ridge dermis, a major dermalepidermal junction, and a pleuristratifed epidermis.Organotypic, or skin equivalent cultures generated with macromolecular crowders can also be used for skin grafting because they have a better developed dermalepidermal junction, which is likely to improve graft success. In addition to enhanced extracellular matrix deposition, macromolecular crowding can be applied to other in vitro systems to enhance reactions with are rate limited, without having to increase the amount of reagents. For example, polymerase chain reactions.

Research

JoVE Journal

Bioengineering

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

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Forschung

Biotechnik

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Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

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