APS wird von Debra International und der British Skin Foundation. University of Dundee Partnerschaft PhD-Programm - Yzn wird von A * STAR unterstützt.

Diese Studie wurde gemäß der Deklaration von Helsinki Grundsätze durchgeführt und wurde von den zuständigen Ethikkommissionen genehmigt. 1. Herstellung von Medien und Reagenzien <ol><li> Vorbereitung der 200x von L-Ascorbinsäure-2-Phosphat-Lager <ol><li> Man löst 29 mg L-Ascorbinsäure-2-phosphat pro 5 ml modifiziertem Eagle-Medium (DMEM) Lösung nach Dulbecco und filtriert über 0,22 um-Membranfilter. Shop als 0,25 ml Aliquots in sterile -20 ° C </li><li> Zugabe von 0,25 ml Aliquot 200x L-Ascorbinsäure-2-phosphat zu jedem Lager 50 ml Fibroblasten-Medium (DMEM mit 1% L-Glutamin und 10% fötalem Rinderserum) am Tag erforderlich ist, für eine Endkonzentration von 0,1 mM L -Ascorbinsäure-2-phosphat. </li></ol></li><li> Herstellung von Keratinozyten-Wachstumsmedium <ol><li> Isolierung und Kultivierung von primären Keratinozyten SCC zuvor 21 beschrieben. Herstellung von Keratinozyten-Wachstumsmedium beschrieben,darin auch. </li><li> Kurz gesagt, bereiten die Medien wie folgt: 300 ml DMEM und 100 ml Ham F-12 mit 10% FBS 0,4 mg / ml Hydrocortison 5 mg / ml Insulin 10 ng / ml EGF 5 mg / ml Transferrin 8,4 ng / ml Choleratoxin 13 ng / ml liothyronine 1x Penicillin-Streptomycin-Lösung </li></ol></li></ol> 2. In-vitro-Konstruktion von Fibroblasten-Mutter Matrix in L-Ascorbinsäure-2-Phosphat ergänzten Medium <ol><li> Saatgut 200000 Fibroblasten pro Well in 6-Well-Platten (20000 Zellen / cm 2) in Fibroblastenmedium mit L-Ascorbinsäure-2-Phosphat ergänzt. Nachspeise alle 2-3 Tage mit 2-5 ml Medium. (Anmerkung: Refeeding Frequenz und Lautstärke eingestellt werden, um die individuellen Bedürfnisse der verschiedenen Zellen angepasst werden Siehe &quot;Diskussionen&quot;.). </li><li> Eine dicke Schicht von Zellen in der extrazellulären Matrix eingebettet wird am Ende von 6 Wochen mit bloßem Auge (bilden <strong&gt; Abbildung 1). Um diese Schicht von der Gewebekulturplatte freigeben, vorsichtig abkratzen den Umfang der Matrix mit einer 1 ml Mikropipettenspitze und drücken Sie dann die Kanten der Matrix zur Mitte des Brunnens. Diese Schicht von Zellen und Matrix (native Matrix) sollte von der Plattenoberfläche leicht lösbar sein, und jetzt werden in den Medien zu schweben. Verbreiten Sie die nativen Matrix, die in den Medien, um zu vermeiden, Klappen auf sich. </li><li> Lassen Sie die native Matrix Schwimmer und renovieren für 5 Tage, verändernden Medien alle 2-3 Tage. Aufgrund Zugfestigkeit und Grenz Umbau in der Matrix wird die Matrix drastisch schrumpfen und sich verringert, um eine kleinere, aber dicker nativen Matrix, und ist bereit, für Invasionsassay verwendet werden. </li></ol> 3. Invasion Assay mit Tumor SCC Keratinozyten <ol><li> Nehmen Sie den nativen Matrix vorsichtig mit stumpfen Pinzette und Transfer zum Nylon Netz. Einmal auf dem Nylon-Netz, verteilt die Matrix vorsichtig, um so flach wie möglich mit einer 1 ml Mikropipette liegenSpitze und stumpfe Pinzette. </li><li> Vorbereitung der sterilen klonalen Zylinder durch Bestreichen einer kleinen Menge von steriler Vaseline an einem Ende. </li><li> Legen Sie die klonalen Zylinder auf dem nativen Matrix, mit der Vaseline Seite nach unten. Dies ist, um eine dichte Abdichtung zwischen der Matrix und den nativen klonalen Ringe gewährleisten. </li><li> Hinzufügen Cscc Zellen an die klonale Zylinder (250.000 Zellen in 100 ul Keratinozyten-Wachstumsmedium). </li><li> Entfernen Sie die klonalen Zylinder nach 6 Stunden, wenn die Cscc Zellen haben sich auf dem nativen Matrix besiedelt. </li><li> Heben Sie die Nylon-Netz mit dem nativen Matrix und Cscc Zellen an der Luft-Flüssigkeit-Grenzfläche auf gebogenen Edelstahl-Drahtgewebe-Unterstützung. </li><li> In Keratinozyten mit Ascorbinsäure ergänzt Wachstumsmedien, bis die Medien-Ebene den Boden des nativen Matrix berührt. </li><li> Ändern Medien alle 2-3 Tage und die Ernte bei 7 und 14 Tage nach der Aussaat von Cscc Zellen. </li></ol> 4. Die Ernte der Kulturen und 3D-Vorbereitung für Histologie <ol><li> Fix in 4% paraformaldehyde über Nacht bei Raumtemperatur. </li><li> Halbierung der Proben und einbetten in Wachs für Formalin-fixierten Paraffin eingebettete Blöcke, mit der Schnittfläche nach außen. </li><li> Alternativ in OCT eingebettet werden die Proben halbiert und Snap-freeze sofort in flüssigem Stickstoff gefrorenes Frischgewebeblöcke. </li><li> 4 um-Schnitte geschnitten auf Mikrotom und Entwachsen (falls erforderlich). Färben mit Standard Hämatoxylin und Eosin. Um die Cscc Zellen sichtbar, monoklonalen Maus-Antikörper gegen Keratin 14 (LL001, in-house) in Immunhistologie Färbung verwendet werden. </li></ol>

<ol>
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Die Autoren haben nichts zu offenbaren.

Aufgrund der Natur des Experiments kann die Gesamtzeit für die Durchführung notwendig bis zu zwei Monaten. Während dieser Zeit müssen größter Sorgfalt und sterile Gewebekulturpraktiken eingesetzt werden, um mikrobielle Kontamination zu verhindern. Abgesehen von ihrer Rolle als Cofaktor bei der Synthese von Hydroxyprolin und Hydroxylysin von Kollagenen, Kollagen stimuliert Ascorbinsäure spezifischen mRNA-Expression in Fibroblasten 22. Die Ascorbin...

Die Verwendung von Biomaterial in 3D-Gewebekultur Forschern ermöglicht, das Verhalten der Zelle im Labor unter physiologischen Bedingungen eher zu einer in vivo-Umgebung als der eines mit 2D Haftung und einem Kunststoffsubstrat rekapituliert studieren. Insbesondere große Fortschritte in der Modellierung mehrschichtigen Epithelien mit der Annahme der 3D-Kulturmethoden an der Luft-Flüssigkeit-Grenzfläche 1-4 gemacht. Solche Techniken getreu nachahmen Keratinozyten-Differenzierung und Tumorzellinvasion ermöglicht größere Flexibilität und Treue für Forscher untersuchen diese Prozesse. Die Wahl der Biomaterial-Substrat, um die Umwelt zu imitieren Stromatumoren wurde in erster Linie die Verwendung von Kollagen Typ I, Engelbreth-Holm-Swarm-Maus-Sarkom-Matrix-und de-epidermized Dermis beteiligt. Zum Beispiel haben Krebs assoziierten Fibroblasten gezeigt, in Richtung der Krebsinvasion 5, Initiierung und Progression über Epithel-Stroma-Wechselwirkungen beitragen 6,7 wohinn in solchen Substraten gewachsen. Der Goldstandard für die Nachahmung der Stroma-Umgebung, in der Haut, die größten und am häufigsten untersuchten geschichteten Epithelien mit solchen Techniken wird betrachtet, um de-epidermized werden menschlichen Dermis (DED). Vorbereitung des DED beinhaltet die Entfernung der Epidermis über Trypsinierung oder physische Abgrenzung von menschlichen kadaver Haut 3,4. Jedoch kann der Zugriff auf solche Haut sehr schwierig für Laboratorien nicht mit klinischen Einrichtungen zugeordnet sind, und erkrankten Dermis ist fast unmöglich zu erzielen. Als Alternative, Labors verwenden häufig eine Kombination aus Typ I-Kollagen (aus Rattenschwänzen isoliert) und / oder Engelbreth-Holm-Swarm-Maus-Sarkom-Matrix. Nach der Entdeckung im Jahr 1927 von 8 Nageotte diesem Kollagen kann leicht mit Essigsäure und Salzfällung isoliert werden, wurde ihre Anwendung auf Gewebekultur anschließend von Huzel Pionierla und Kollegen 9. Collagen Beschichtung erwies sich als überlegen zu Glas für die Zellkultur von 29 Stämmen und Gewebeexplantaten wie von Ehrmann und Gey 9 verhört zu sein. Derzeit ist der Haupttyp von Kollagen in der Gewebekultur verwendet, die aus Rattenschwanzsehnen isoliert und wird üblicherweise von kommerziellen Quellen gekauft werden. Allerdings ist der Nachteil für treue Substrat Reprise, dass der Rattenschwanz Kollagen ist nicht identisch mit dem menschlichen Kollagen oder die menschlichen Dermis, wobei Typ I und III Kollagen als Hauptbestandteile vorhanden sind, und isolierten Rattenschwanz Kollagen ist immer fragmentiert. Engelbreth-Holm-Swarm-Maus-Sarkom-Matrix ist eine gallertartige Proteingemisch durch kultivierte Engelbreth-Holm-Swarm-Maus-Sarkom-Zellen 10 abgesondert. Die Hauptbestandteile sind Laminin, Kollagen Typ IV, Heparinsulfat-Proteoglykan, Entactin und Nidogen und die genauen Verhältnisse von diesen Proteinen wird von Charge zu Charge variieren. Abgesehen von strukturellen ProProteine ​​enthält diese Matrix auch signifikante Mengen an Wachstumsfaktoren, wie den transformierenden Wachstumsfaktor β, epidermaler Wachstumsfaktor, insulinähnlichen Wachstumsfaktor 1, Rinder-Fibroblasten-Wachstumsfaktor und von Blutplättchen abgeleiteten Wachstumsfaktor, Zellverhalten 11,12 verändern würde. Zeigt auf die schiere Komplexität des Engelbreth-Holm-Swarm-Maus-Sarkom-Matrix, wurden insgesamt 1.851 Proteine ​​in einer aktuellen Studie 13 Proteom identifiziert. Angesichts der reichen und komplexen Natur dieser Matrix ist Vorsicht wurde bei der Auslegung und Vergleich verschiedener Experimente mit dem Einsatz von IT-11 beraten. Unsere Labors haben ein großes Interesse in der genetischen Hauterkrankungen, insbesondere solche mit einer Prädisposition zur Entwicklung von kutanen Plattenepithelkarzinomen (Cscc) 14. Im Fall der rezessiven dystrophischen Epidermolysis bullosa (RDEB), einer schweren Erkrankung mit Blasenbildung Keimbahnmutationen im COL7A1-Gen <sbis&gt; 15 bis 17 haben wir festgestellt, dass die dermale Mikroumgebung bei diesen Patienten tumorfördernde 18. Im Verlauf der Studie konnten wir den Tumor fördernden Eigenschaften von Hautfibroblasten in Kollagen eingebettet I / Engelbreth-Holm-Swarm-Maus-Sarkom-Matrix und untersucht Möglichkeiten zur Bewertung von zelleigenen, nativen Matrix beurteilen. Um dies zu erreichen, modifizierten wir eine vorherige Technik aus dem Labor von Lucie Germain arbeitet auf der menschlichen Haut Äquivalente 19,20. Germain Technik konnte die menschliche Haut mit gut organisierten Basalmembran mit primären humanen Keratinozyten und Fibroblasten-Kulturen in Abwesenheit von einem synthetischen Gerüst oder Kadaver rekonstruieren. In diesem Beitrag werden die verwendet, um die Haut-Tumor Stroma-Mikroumgebung (native Matrix) rekapitulieren Schritte von primären stromalen Fibroblasten in vitro direkt abgeleitet werden beschrieben 18. Ureinwohner Matrizen pdurch langfristige Kultur von Fibroblasten roduced wurden als dermales Äquivalent zum Assay für Cscc Zellinvasion verwendet. Wir mit nativen Matrix entweder aus der extrazellulären Matrix durch RDEB Fibroblasten (bei Typ-VII-Kollagen (C7)-Mangel) oder von Fibroblasten RDEB retroviral mit einem Kollagen-Typ-VII zum Ausdruck zu konstruieren und zeigen die tiefgreifende Wirkung einer einzelnen Kollagen auf Tumor transduzierten abgesondert abgeleitet präsentieren Daten Zellinvasion.

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			<td class="xl63" height="64" style="height:48.0pt;width:66pt" width="88">L-Ascorbic acid 2-phosphate</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">A8960</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl65" height="42" style="height:31.5pt;width:66pt" width="88">DMEM with l-glutamine,</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">Life Technologies</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">11995-073</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
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		<tr height="106" style="height:79.5pt">
			<td class="xl63" height="106" style="height:79.5pt;width:66pt" width="88">4,500 mg/L d-glucose, 110 mg/L sodium pyruvate</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl63" height="85" style="height:63.75pt;width:66pt" width="88">100x Penicillin-Streptomycin</td>
			<td class="xl63" style="width:66pt" width="88">Life Technologies</td>
			<td class="xl63" style="width:66pt" width="88">15070</td>
			<td class="xl64" style="width:66pt" width="88"></td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Vaseline</td>
			<td class="xl63" style="width:66pt" width="88">VWR</td>
			<td class="xl63" style="width:66pt" width="88">PROL28908.290</td>
			<td class="xl64" style="width:66pt" width="88"></td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Clonal cylinders</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">Z370789</td>
			<td class="xl64" style="width:66pt" width="88"></td>
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		<tr height="148" style="height:111.0pt">
			<td class="xl63" height="148" style="height:111.0pt;width:66pt" width="88">Nylon Net Filter Disc Hydrophilic 100um 25um diameter 100/pk</td>
			<td class="xl63" style="width:66pt" width="88">Millipore</td>
			<td class="xl63" style="width:66pt" width="88">NY1H02500</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="127" style="height:95.25pt">
			<td class="xl63" height="127" style="height:95.25pt;width:66pt" width="88">Bent stainless steel wire mesh support</td>
			<td class="xl63" style="width:66pt" width="88">Made in house</td>
			<td class="xl63" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88">Dimensions were made so that the mesh would fit into 6-well plates</td>
		</tr>
	</tbody>
</table>

tissue engineering of tumor stromal microenvironment with application to cancer cell invasion

3D organotypischen Kulturen von Epithelzellen auf einer Matrix mit mesenchymalen Zellen eingebettet sind weit verbreitet, um epitheliale Zelldifferenzierung und-invasion zu studieren. Rattenschwanz Kollagen Typ I und / oder Matrix von Engelbreth-Holm-Swarm-Maus-Sarkom-Zellen abgeleitet wurden traditionell als Substrate eingesetzt, um die Matrix oder Stroma-Mikroumgebung, in die mesenchymalen Zellen (in der Regel Fibroblasten) bevölkert modellieren. Obwohl Versuche mit solchen Matrizen sind sehr informativ, kann argumentiert werden, dass wegen eines überwiegenden Vorhandensein eines einzelnen Proteins (wie in Typ-I-Collagen) oder einem hohen Gehalt an Basalmembran-Komponenten und Wachstumsfaktoren (wie z. B. in einer Matrix aus der Maus stamm Sarkom-Zellen), sind diese Substrate nicht spiegeln die besten Beitrag zum Matrix-Zusammensetzung durch die Stromazellen selbst gemacht. Um Mutter Matrizen durch primäre dermale Fibroblasten von Patienten mit einem Tumor isoliert anfällig, genetische Blasenstörung (rezessiven dystrophischen hergestellt studierenEpidermolysis bullosa) haben wir einen bestehenden nativen Matrix-Protokoll, um die Tumorzellinvasion studieren angepasst. Fibroblasten induziert werden, ihre eigene Matrix über einen längeren Zeitraum in Kultur produzieren. Diese nativen Matrix wird dann von der Kulturschale abgelöst und Epithelzellen auf sie ausgesät bevor das gesamte Co-Kultur wird die Luft-Flüssigkeit-Grenzfläche erhöht. Zelluläre Differenzierung und / oder Invasion kann dann über die Zeit beurteilt werden. Dieses Verfahren bietet die Möglichkeit, epitheliale-mesenchymale Zellwechselwirkungen in einer 3D Umgebung, ohne die Notwendigkeit für eine synthetische oder Fremdmatrix mit dem einzigen Nachteil, dass die längere Zeit erforderlich, um die native Matrix zu beurteilen. Die Anwendung dieser Technik beschreiben wir die Fähigkeit eines einzelnen Moleküls von Fibroblasten, Kollagen VII, um die Tumorzellinvasion hemmen beurteilen.

<html> Diese Technik eröffnet die Möglichkeit der Prüfung und Vergleich der invasive Verhalten von Tumorzellen (in diesem Fall Cscc) unter verschiedenen 3D Stromazellen Umgebungen. Mit dieser Technik kann nicht nur C7-defizienten nativen Matrizen, die die Haut RDEB Umwelt rekapituliert erzeugt werden, sondern auch zusätzliche Matrizen, die genetisch zu über-express C7 18 konstruiert wurden. Wie in Abbildung 2 zu sehen, Verletzung der RDEB Cscc Keratinozyten war in C7-Überexpression nativ...

Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Gewebezüchtungen Fibroblasten-nativen Matrix ist eine aufstrebende Werkzeug, um eine Stroma-Substrat, das epitheliale Zellproliferation und Differenzierung unterstützt generieren. Hier wird ein Protokoll gemäß dieser Methode die Auswirkung verschiedener stromalen Zelltypen auf Tumor Zellbiologie Beurteilung vorgelegt.

Tissue Engineering von Tumor-Stroma-Mikromilieu mit Anwendung auf die Invasion von Krebszellen

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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Research

JoVE Journal

Bioengineering

9.7K Aufrufe.  University of Dundee.  Gewebezüchtungen Fibroblasten-nativen Matrix ist eine aufstrebende Werkzeug, um eine Stroma-Substrat, das epitheliale Zellproliferation und Differenzierung unterstützt generieren. Hier wird ein Protokoll gemäß dieser Methode die Auswirkung verschiedener stromalen Zelltypen auf Tumor Zellbiologie Beurteilung vorgelegt. 

Serie: Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

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

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Directed Cellular Self-Assembly to Cell-Derived Tissue Ringe für biomechanische Analyse und Tissue Engineering Fabricate

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Dreidimensionale Zellkultur-Modell zur Messung der Auswirkungen der interstitiellen Flüssigkeit Flow auf Tumorzellinvasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineering Skeletal Muscle Gewebe von Murine Myoblast Progenitor Cells und Anwendung von Elektrostimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering eines Menschen 3D<em&gt; In-vitro-</em&gt; Tumor-Testsystem

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Ingenieurwesen Fibrin-basierten Tissue Konstrukte von Myofibroblasten und Anwendung von Constraints und abseihen to Cell und Collagen (Re) Organisation Induce

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Langzeit-Intravitalimmunfluoreszenz-Imaging von Gewebematrix Bestandteile mit Epifluoreszenz und Zwei-Photonen-Mikroskopie

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Sandwichartigen Mikroumgebungen zu nutzen Handy / Material-Interaktionen

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

In-vitro-Modell von physiologischen und pathologischen Blutfluss mit Anwendung auf Untersuchungen der Gefäßzell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Tissue Engineering von Intrinsic Vaskularisation in ein<em&gt; In Vivo</em&gt; Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...