Ein. Gründung der Fibroblasten-Kulturen aus Hautexplantaten <ol><li> 4 mm Stanzbiopsien vom menschlichen Unterarm erworben werden in MEM platziert ergänzt 100 Einheiten / ml Penicillin, 100 ug / ml Streptomycin und 0,25 pg / ml Fungizone. </li><li> Schneiden Sie subkutanes Fett und fein hacken die Biopsie in kleine Stücke durch Schaukeln eine Reihe 24 Skalpell gegen den Boden der Petrischale. </li><li> Die Gewebeschnitte Aufschlämmung in einem 25 cm 2 Gewebekulturbehälter mit primären Fibroblasten Wachstumsmedium (MEM mit 10% FCS, 100 Einheiten / ml Penicillin, 100 ug / ml Streptomycin und 25 ug / ml Fungizon ergänzt) zugesetzt, bis die Oberfläche des Fadens bedeckt, aber flüssige Tiefe unzureichend für Gewebe zu schweben. </li><li> Nach 3-tägiger Inkubation bei 37 ° C in einer befeuchteten Atmosphäre von 5% CO 2, 3 ml von primären Fibroblasten-Wachstumsfaktor Medien. </li><li> Nach einer weiteren 3 Tagen Inkubation Change-Medien mit frischen primären Fibroblasten-Wachstumsfaktor Medien, oder wenn cells sind nahezu konfluenten sie 1.04 kann in frischen Medien aufgeteilt werden. (Fungizone können ab diesem Zeitpunkt entfallen). </li></ol> 2. Stufe I - Herstellung von Kollagen I aus Rattenschwänzen Hinweis: Protokoll für ca. 12-14 Jugendlichen (frisch oder gefroren) Rattenschwänzen <ol><li> Planen Rattenschwanz durch Waschen in 70% igem Ethanol und entfernen Sehnen wie folgt: </li><li> Entfernen Sie die Haut vor Rattenschwanz durch Schneiden in der Mitte des Schwanzes von oben nach unten mit einem Skalpell und Läuten entlang der Länge des Schwanzes. </li><li> Trennen Sehne vom Kern des proximalen Bereich des Schwanzes. </li><li> Entfernen Sehne zu dem distalen Bereich des Schwanzes Vermeidung der Hülle mit gezahnten Pinzette. </li><li> Extrakt 1 g Sehne / 250 ml 0,5 M Essigsäure durch Rühren bei 4 ° C für 48 h durchgeführt. </li><li> Zentrifugieren Extrakt (7.500 xg) für 30 Minuten und entsorgen Sie die Pellets. </li><li> Hinzufügen eines gleichen Volumens von 10% (w / v) NaCl zu dem Überstand, und rühren für 30-60 min. </li><li&gt; Säule (10.000 × g) für 30 Minuten, den Überstand verwerfen und wieder aufzulösen der Niederschlag in 0,25 M Essigsäure bei ~ 1:1-Verhältnis von Rühren für 24 h bei 4 ° C. </li><li> Dialysieren das Kollagen Lösung gegen 6-8 Änderungen 6L ~ 17,5 mM Essigsäure (1 ml Essigsäure je Liter kaltes Wasser, Wechsel 2 x täglich). </li><li> Zentrifuge dialysiert Kollagen bei 30.000 xg für 1,5 Stunden. </li><li> Überstand entfernen und in sterile Flasche. </li><li> Einstellen der Kollagen I-Konzentration auf ~ 2 mg / ml unter Verwendung von 0,5 mM Essigsäure bei 4 ° C. </li></ol> 3. Stage II - Einrichten der 3D-Matrix mit eingebetteten Fibroblasten (lassen für 8 Tage kontrahieren). <ol><li> Montieren Sie die Mischung mit kalter Reagenzien bei 4 ° C in einer vorgekühlten Flasche. Halten Kollagen I auf Eis. </li><li> Für ein T75-Flasche von konfluenten Fibroblasten (~ Zellzahl 1 x 10 6) Verwendung: <ul><li> 25 ml Rattenschwanz Kollagen (ca. conc 2mg/ml) </li><li> 3 ml 10xMEM </li><li> 0,22 M NaOH: erste, fügen 2ml, dann tropfenweise unter Rühren, bis das Kollagen wird orange, </li><li> aber nicht rosa (in der Regel bis zu 3 ml). Ungefähre p H = 7,2. </li><li> Anmerkung: Es ist wichtig sicherzustellen, dass die Mittel / Gel-neutral oder schwach sauer bleibt als Fibroblasten nicht zusammenziehen wird, wenn das Gel noch leicht alkalischen Bedingungen ausgesetzt. </li></ul></li><li> Trypsinise die Fibroblasten, drehen bei 400 xg für 5 Minuten und entfernen Überstand. </li><li> Während 5 Minuten vor spinnen: Bereiten 12 x 35 mm Kunststoff-Geschirr - normalerweise erreicht 12 Gerichte aus einem Kolben von Fibroblasten / Kollagen-Mix. </li><li> Resuspendieren Fibroblasten in 3 ml FCS und sofort an das Kollagen Mischung hinzufügen und umrühren. </li><li> Platte etwa 2,5 ml Kollagen / Fibroblasten pro Schale so schnell wie möglich. Versuchen Sie, Blasen und Kollagen Einstellung zu vermeiden. </li><li> Lassen Sie das Kollagen bei 37 ° C in einer befeuchteten Atmosphäre von 5% CO 2 in Luft für 10 Minuten eingestellt. </li><li> 1ml von Fibroblasten wachsenth (DMEM + 10% FCS). </li><li> Trennen der Kollagen / Fibroblasten-Matrix von den Seiten der Schale mit Hilfe einer Pipette. </li><li> Nächster Tag: In 1 ml Fibroblasten-Wachstumsfaktor (DMEM + 10% FCS). </li><li> Ändern Medien jeden zweiten Tag. Lassen Kollagen / Fibroblasten Matrix Vertrag für ca. 8 Tage, bis sie in einer 24-Well-Platte passen (Kontraktion von ~ 3,5 cm bis 1,5 cm im Durchmesser, siehe Abbildung 1). </li></ol> Hinweis: Die Kollagen-Konzentration sollte so eingestellt werden, um die Anwendung angepasst werden. Je verdünnter die Kollagenlösung, desto schneller wird es durch die Fibroblasten kontrahiert werden. Geringfügige Unterschiede in Kontraktionsrate wird mit verschiedenen Chargen von Kollagen in der gleichen Konzentration erfahren werden. Ebenso verschiedenen Fibroblasten-Kulturen wird die Kollagen-Gele mit unterschiedlichen Geschwindigkeiten kontrahieren, und je mehr Fibroblasten präsentieren, desto schneller die Geschwindigkeit der Kontraktion. Kollagenkonzentration und Fibroblasten-Dichte kann daher durch angepasst, um die Rate der Kontraktion zu verändern,und die endgültige Dichte des Kollagengels. 4. Stage II - Plating Zellen von Interesse auf der Oberseite der Matrix Hinweis: Sterilisieren alle Zangen und Geräte mit Ethanol vor dem Gebrauch. <ol><li> Mit stumpfen Pinzette vorsichtig bewegen vertraglich Matrix 24-Well-Schale. Stellen Sie sicher, dass es nicht klappt. </li><li> Bereiten Suspension von Zellen von Interesse bei etwa 4 x 10 4 / ml und Platte 1ml (nach trypsinising, Spin-Zellen loszuwerden Trypsin) auf der Oberseite der Matrix. Tatsächliche Anzahl der Zellen benötigt variiert je nach Zelltyp. Zellmedium sollte normalen Wachstumsmedium für die Zellen von Interesse sein. </li><li> Erlauben Zellen bis zur Konfluenz auf der Matrix, etwa 3-5 Tage wachsen gelassen. </li></ol> 5. Stufe III - Übertragung der Matrix auf einem Raster für die Invasion (ca. 0-21 Tage) <ol><li> Cut Edelstahlgitterroste ein Stativ zu schaffen und autoklavieren vor der Anwendung (siehe Abbildung 2). </li><li> Zeigen sterile Gitter6 cm Schale, fügen Wachstumsmedium bis zu einem Niveau oberhalb des Gitters (ca. 10.5ml). Matrix Platz auf dem Gitter und sanft absaugen Medien so dass die Unterseite der Matrix in Kontakt mit den Medien, aber nicht getaucht. Dies wird als die Luft / Flüssigkeits-Schnittstelle, die einen Gradienten, Invasion fördert schafft bezeichnet. Ersetzen Medium alle zwei Tage (siehe Abbildung 2). </li><li> Live-Zelle kann kontextuellen Bildgebung mit fluoreszierenden Zellen zu diesem Zeitpunkt oder früher abgebildet werden. Die kontrahierten oder fibrillären Kollagen kann ich abgebildet mit Second Harmonic Generation (SHG, siehe Abbildung 3). Mittels Multi-Photonen-Anregung mit großem Feld Detektion kombinierten wir feststellen, dass SHG mindestens 100 um in die Matrix abgebildet werden, wohingegen Zellen, die GFP cytoplasmatischen abbildbar mindestens 200 um tief sein. </li></ol> Hinweis: Im Hinblick auf die Quantifizierung der Invasion, definiert der Tag, an dem Matrizen auf den Gittern platziert werden Tag 0. Platzierung auf Gittern einen Gradienten von Zellkulturmedien, die Invasion int fördert<html> o die Matrix. Die Proben können in den nächsten 1 abgebildet werden - 21 Tage (oder länger) an biologischen Prozessen wie Invasion, Proliferation, Überleben oder Differenzierung (siehe Referenzliste) zu beurteilen. 6. Stufe IV - Fixation <ol><li> 5 ml 4% PFA in einem Falcon-Röhrchen. </li><li> Übertragen Sie die Matrix auf eine ebene Fläche. Schneiden Sie die Matrix in zwei Hälften mit einem frischen, sauberen Skalpell (wie werden Sie Färbung der Querschnitt), heben Sie die Matrix mit Skalpell und Transfer zum 4% PFA, fix über Nacht. </li><li> Organotypische Matrizen sind nun bereit, mit dem Antikörper färben / Fleck der Wahl (siehe Abbildung 3). </li></ol> 7. Repräsentative Ergebnisse: <img alt="Abbildung 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> Abbildung 1 Beispiel Fibroblasten vergeben Kollagen I. Mischung aus Fibroblasten und Rattenschwanz Kollagen aus der Petrischale gelöst und erlaubt, über 6-8 Tage schrumpfen. Abbildung 2 &quot;src =&quot; / files/ftp_upload/3089/3089fig2.jpg &quot;/&gt; Abbildung 2 Organotypische Assay Progression. Eine schematische organotypischer Einrichtung und Progression. B, Beispiel von Kollagen / Fibroblasten-Matrix auf der Oberseite Edelstahl, sterilen Gitters, um Luft / Flüssigkeits-Grenzfläche zu schaffen. <img alt="Abbildung 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> Abbildung 3 Anwendungen organotypischer Assay. A, B eindringenden nichtinvasiven und invasiven duktalen Adenokarzinom (PDAC) Zellen im Laufe der Zeit mit organotypischen Matrix. C, lebenden Hautäquivalent, das eine geschichtete Epidermis eines Fibroblasten vergebene kollagenen dermalen Komponente. D, eindringenden C8161 Melanomzellen in einem Fibroblast -Vertrag kollagenen dermal gleichwertig. E, invasive PDAC Zellen (grün) interagieren und Invasion mit Fibroblasten (rot) in der organotypischen Matrix. F </strong&gt;, visualisiert invasive PDAC Zellen (grün), die mit einer entwürdigenden Umgebung extrazellulären Matrix (violett) mit Multiphotonen-basierte Erzeugung der zweiten Harmonischen.

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

Wir haben nichts zu offenbaren.

Hier stellen wir ein Verfahren zur Herstellung einer 3-dimensionalen Matrix geeignet für Untersuchungen von invasiven Zellmigration 6-8. Die Matrix besteht aus Kollagen Typ 1 Fibrillen, die sich über einen Zeitraum von mehreren Tagen durch primäre menschliche Fibroblasten epidermalen zusammengezogen sind. Das Kollagen durch Säureextraktion nicht enzymatische Verdauung, die Reaktivität bewahrt an den Poly-Peptid Enden und fördert Vernetzung der Kollagenfibrillen zu größeren Aggregaten bei Neutralisation der Pufferbedingungen 9 hergestellt. Dies führt zu einem rekonstituierten Gel, stärker imitiert die Funktionen von Kollagen in vivo und hat wichtige Konsequenzen für die invasive Eigenschaften von Zellen in den Gelen gezüchtet. Es spielt keine Rolle, ob frisch oder gefroren Ausgangsmaterial verwendet wird, aber die Verwendung von Jugendlichen Rattenschwänzen ist wichtig, weil Kollagen Vernetzung ist labile bei jüngeren Tieren. Collagen I von jüngeren Tieren ist daher leichter zu extrahieren und re-stellt with höhere Treue. Die Kollagenmatrix fixiert und mit Antikörpern gegen entweder invasiv Tumorzellen oder Stromafibroblasten 2,10 gerichteten gefärbt werden und sind sehr nützlich zum Testen der Wirksamkeit von Wirkstoffen, die Invasion 5,6 hemmen könnte. Obwohl dieses Protokoll schlägt die Verwendung von primären humanen Hautfibroblasten, ist es machbar, primäre Fibroblasten, die von anderen Gewebearten zu verwenden, je nach der Art der zu untersuchenden Gewebe. Es ist auch möglich, die Kulturen mit immortalisierten Fibroblasten, einschließlich Zellen, die stabil mit fluoreszierenden Proteins transfiziert Konjugate herzustellen. Auf diese Weise sowohl Stroma-und Tumorzellen markiert, um Zell-Zell-Interaktionen während der Invasion 11 visualisieren.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> 10x MEM </td><td> Gibco </td><td> 21430 </td><td> - </td></tr><tr><td> NaOH </td><td> Sigma </td><td> 367.176-500G </td><td> Bereiten 0,22 M Vorratslösung in Wasser </td></tr><tr><td> FCS </td><td> PAA Laboratories </td><td> A15-101 </td><td> - </td></tr><tr><td> 35 mm Schalen </td><td> Falke </td><td> 353001 </td><td> Für den Schritt 3,4 </td></tr><tr><td> 60 mm Schalen </td><td> Falke </td><td> 353004 </td><td> Für den Schritt 5,2 </td></tr><tr><td> Frühling Pinzette stumpf </td><td> Samco </td><td> E003/02 </td><td> Zahnriemen, nicht glatt </td></tr><tr><td> 16% Paraformaldehyd </td><td> Elektronenmikroskopie Dienstleistungen </td><td> 15710 </td><td> Verdünnte bis 4% in PBS vor der Verwendung </td></tr><tr><td> Bildschirme für cd-1, Größe 40 mesh </td><td> Sigma </td><td> S07707-5EA </td><td> Edelstahlgitterroste, 5 pro Packung </td></tr><tr><td> Dialyseschlauch </td><td> Medicell International </td><td> 7607 2295 </td><td> 12 bis 14 kD </td></tr><tr><td> PBS </td><td> Oxoid </td><td> BR0014G </td><td> - </td></tr><tr><td> Essigsäure </td><td> Sigma </td><td> 242853 </td><td> - </td></tr><tr><td> 24 Well-Platte </td><td> Falke </td><td> 353047 </td><td> Für den Schritt 4,1 </td></tr><tr><td> Fungizone </td><td> In Vitrogen </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Zellmigration ist grundlegend für viele Aspekte der Biologie, einschließlich Entwicklung, Wundheilung, die zellulären Reaktionen des Immunsystems und Metastasierung von Tumorzellen. Migration hat auf Deckgläsern wurden untersucht, um zelluläre Dynamik zugänglich Untersuchung mit dem Lichtmikroskop zu machen. Allerdings hat sich gezeigt, dass viele Aspekte der Zellmigration auf Merkmale der lokalen Umgebung einschließlich seiner Elastizität Proteinzusammensetzung und Porengröße, die nicht originalgetreu durch starre zweidimensionalen Substraten wie Glas und Kunststoff 1 dargestellt abhängen. Darüber hinaus hat die Interaktion mit anderen Zelltypen, einschließlich Stromafibroblasten 2 und Immunzellen 3, wurde gezeigt, dass eine kritische Rolle bei der Förderung der Invasion von Krebszellen spielen. Untersuchung auf molekularer Ebene zunehmend gezeigt, daß molekularer Dynamik, einschließlich Reaktion auf Medikamente, identischer Zellen signifikant verschieden sind, wenn verglichen <em&gt; In vitro und in vivo 4. Im Idealfall wäre es am besten, um die Zellwanderung in seiner natürlich vorkommenden Kontext, in lebenden Organismen zu studieren, dies ist jedoch nicht immer möglich. Zwischenprodukt Gewebekultursystemen wie Zellen abgeleiteten Matrix, Matrigel, organotypische Kultur (hier beschrieben) Gewebeexplantate, Organoiden und Xenotransplantate sind daher wichtig experimentellen Zwischenprodukte. Diese Systeme ungefähre bestimmte Aspekte der in vivo-Umgebung sind aber offen für experimentelle Manipulation wie die Verwendung von stabil transfizierten Zelllinien, medikamentöse Behandlung Regime, langfristige und hochauflösende Bildgebung. Solche Zwischensysteme sind insbesondere als Prüfgeländen um Sonden herzustellen validieren und Parameter, die zur Abbildung des dynamischen Verhaltens von Zellen und fluoreszierenden Reporter vor der Durchführung Bildgebung in vivo 5. Als solche können sie eine wichtige Rolle bei der Verringerung des Bedarfs an Experimente dienen living Tieren.

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Es wird ein Verfahren zur Herstellung einer 3-dimensionalen Matrix, bestehend aus Kollagen Typ I und primären humanen Fibroblasten beschrieben. Diese organotypischen Gel dient als nützliches Substrat invasive Zellmigration beurteilen, da es grundlegende Funktionen von Gewebestroma nachahmt und ist offen für viele Formen der Mikroskopie.

Organotypische Collagen I Assay: A Temperguss Platform to Cell Verhalten in 3-Dimensional Kontext beurteilen

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

organotypic-collagen-i-assay-malleable-platform-to-assess-cell

Research

JoVE Journal

Bioengineering

22.3K Aufrufe.  University of Glasgow. Es wird ein Verfahren zur Herstellung einer 3-dimensionalen Matrix, bestehend aus Kollagen Typ I und primären humanen Fibroblasten beschrieben. Diese organotypischen Gel dient als nützliches Substrat invasive Zellmigration beurteilen, da es grundlegende Funktionen von Gewebestroma nachahmt und ist offen für viele Formen der Mikroskopie.

Serie: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

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 ...

Forschung

Biotechnik

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Kultivierung humaner neuronaler Vorläuferzellen in einer 3-dimensionalen Self-Montage Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Selbstberichts Gerüste für 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Ein verbessertes Verfahren zur Herstellung von Typ I-Kollagen aus der Haut

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analyse der Zellmigration in eine dreidimensionale Kollagenmatrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Gradient Diffusionssystem, um Chemotaxis in einem 3D-Kollagen-Matrix Untersuchen

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Zerkleinerte Gewebe in komprimierter Collagen: Ein zellhaltigen Biotransplant für einstufige rekonstruktive Reparatur

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Rekombinante Kollagen Modell I Peptid-Microcarriers für Zelle Expansion und ihre mögliche Verwendung als Zelle-Delivery-System in einem Bioreaktor

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.