1. Assay Set-up <ol><li> Auftauen einer kleinen aliquoten (&lt;500 ul) von Matrigel auf Eis bei 4 ° C (etwa 2 Stunden). </li><li> Bereiten Sie Gel-Rezeptur (siehe Beispiel Volumina in Tabelle unten): 10x PBS (1x im Gesamtvolumen), 1 N Natronlauge (entspricht 0,023 Bände hinzugefügt Kollagen, respektive dem Kollagen den Empfehlungen des Herstellers, soweit erforderlich), geben Sie Matrigel und Kollagen I zu Endkonzentrationen von 1 mg / ml und 1,3 mg / ml (andere Matrix-Formulierungen verwendet werden, je nach Zelltyp und Experiment werden). </li></ol> Beispiel Gel-Rezeptur <table border="1"><tbody><tr><td> Komponenten </td><td> Stoffkonzentration </td><td> Endkonzentration </td><td> Fügen Volumen </td></tr><tr><td> 10X PBS </td><td> 10X </td><td> 1X </td><td> 0,090 ml </td></tr><tr><td> Steriles Wasser </td><td></td><td></td><td> 0,346 ml </td></tr><tr><td> 1 N NaOH </td><td></td><td></td><td> 0,008 ml </td></tr><tr><td> Matrigel </td><td> 9,90 mg / ml </td><td> 1 mg / ml </td><td> 0,101 ml </td></tr><tr><td> Rattenschwanz Kollagen Typ I </td><td> 3,66 mg / ml </td><td> 1,3 mg / ml </td><td> 0,355 ml </td></tr></tbody></table><ol start="3"><li> Mischen Komponenten des Gels auf Eis in der gleichen Reihenfolge wie oben und inkubieren endgültige Lösung für 1 Stunde bei 4 ° C Nach unserer Erfahrung, eine 1-stündigen Inkubation vor dem Aussähen der Zellen führt zu einer gleichmäßigeren Gelierung des Kollagens. Achten Sie darauf, Matrigel und Kollagen auf Eis halten zu allen Zeiten und an so schnell wie möglich arbeiten, um die Gelierung zu verhindern. </li><li> Platz 12 mm Durchmesser 8 um Pore Zellkultur-Inserts in eine 12-Well-Platte mit einer sterilisierten Pinzette. </li><li> Zählen von Zellen und Resuspendieren in serumfreiem Medium bei 5 × 10 6 Zellen / ml (Gesamtvolumen sollte 10% des Gel-Lösung). </li><li> Geben Sie 100 ul Zell-suspension zu 900 ul Gel-Lösung (Endzellkonzentration 5 x 10 5 Zellen / ml) mischen und gründlich durch Pipettieren vorsichtig nach oben und unten. </li><li> Fügen Sie 150 ul der fertigen Mischung für jeden Einsatz und Transfer zu einem 37 ° C, 5% CO 2-Inkubator für 30 Minuten, bis Gel polymerisiert. </li><li> Mit Serum-freien Medien, </li></ol><ul><li> Geben Sie 100 ul oben auf dem Gel und 1200 ul unter dem Einsatz für den statischen Zustand. Die Flüssigkeitsstand im Inneren des Einsatzes und außen in der auch etwa gleich sein sollte, was zu einer minimalen Druckdifferenz über dem Gel und ohne interstitielle Strömung. </li><li> Geben Sie 100 ul unter dem Einsatz und 650 ul oben Gel für den Strömungszustand. Seien Sie vorsichtig, um mögliche Luftblasen unterhalb des Einsatzes zu vermeiden, da diese Zellen, die aus der Migration durch die Membran an diesen Standorten zu verhindern. Die Druckdifferenz, die dieses Volumen erzeugt wird, ungefähr 1,3 cm H 2 O (oder 1 mm Hg). </li></ul><ol start="9"><li> Legen Sie die Platte in einem 37° C, 5% CO 2-Inkubator für 24 Stunden. </li></ol> 2. Zellfärbung und Zählen <ol><li> Fügen Sie 500 ul 1X PBS pro Vertiefung in neue 24-Well-Platte, das wird benutzt, um die Einsätze zu waschen. </li><li> Entfernen des Mediums restlichen in dem oberen Abschnitt der Strömung Transwells und die Lautstärke festzulegen. Berechnen Sie insgesamt eluierten Volumen durch Subtraktion des verbleibenden Volumens aus dem Gesamtvolumen ursprünglich aufgenommen, 650 ul (dies wird für Durchfluss Berechnungen verwendet werden, siehe unten). Wattestäbchen, um das Gel aus den Einsätzen zu entfernen und um die obere Oberfläche der Membran Wischtuch nicht befallenen Zellen zu entfernen. Legen Sie die Einsätze in den 24-Well-Platte mit 1X PBS für 15 s, um Einsätze zu waschen. </li><li> PBS entfernen und hinzufügen 500 ul von 4% Paraformaldehyd (PFA) unter jedem Einsatz, Inkubation 30 Minuten bei Raumtemperatur, um die transmigrierten Zellen zu fixieren. </li><li> Entfernen Sie die PFA und spülen Sie einmal mit 500 ul 1X PBS, um restliches Fixativ entfernen. </li><li> Fügen Sie 500 ul of 0,5% Triton X-100-Lösung unter den Einsätzen und inkubieren für 10 Minuten bei Raumtemperatur, um die Zellen zu permeabilisieren. </li><li> Schneiden Sie aus Membranen des Einsatzes mit einer Rasierklinge und Ort in 100 ul 2 pg / ml DAPI in 1X PBS-Lösung, wobei darauf geachtet, um die Unterseite der Membran der Vorderseite nach unten platzieren (transmigrierten Zellen nach unten). </li><li> Wrap Platte in Alufolie nehmen und auf einem Schüttler bei 150 rpm für 30 Minuten bei Raumtemperatur. </li><li> Waschen Sie Membranen in 500 ul 1X PBS auf Schüttler (3 Mal wiederholen für jeweils 10 Minuten), um freie DAPI zu entfernen. </li><li> Ort Membranen auf Glasplättchen mit transmigrierten Zellen nach oben, fügen Montagelösung und Deckglas. </li></ol> 3. Datenanalyse <ol><li> Count DAPI-gefärbten Zellkernen auf 5 zufällig ausgewählten Standorten jeder Membran (bleiben Sie weg von den Rändern und verwenden Sie einen 10x-oder 20x Objektiv). </li><li> Berechnen des Durchschnitts-Zellen und erhalten die prozentuale Invasion durch Verwendung der folgenden Formel: Invasion Prozent = 100% x (durchschnittliche Zellzahl x Membranfläche) / (Fläche des mikroskopischen Bildes x Anzahl der Zellen seeded) Die Invasion Prozent bis zu einem gewissen Kontrollbedingung (in der Regel der statische Zustand) um einen Vergleich zwischen unabhängigen Experimenten ermöglichen normalisiert werden. </li><li> Berechnen der durchschnittlichen Fließgeschwindigkeit: Die Gesamthöhe eluierte Volumen in der Strömungszustand von der Inkubationszeit (z. B. 24 Stunden). </li><li> Berechnen Sie die mittlere Fließgeschwindigkeit: Teilen Sie die mittlere Strömungsgeschwindigkeit durch die Querschnittsfläche des Gels / Membran (in diesem Fall 60 mm 2). </li></ol> 4. Repräsentative Ergebnisse Um Tumorzellinvasion unter interstitielle Strömung zu messen, führten wir unsere 3-D-Flow Invasion Assay unter Verwendung von MDA-MB-435S metastasierendem Melanom-Zellen. Diese Zellen haben früher gezeigt, dass in Reaktion auf interstitielle Fluidströmung 13-14 einzudringen. Die Zellen wurden in einer Matrix von 1,3 mg / ml Ratten-tai aus eingebettetenl Sehne Kollagen Typ I und 1 mg / ml Matrigel Basalmembran-Matrix in einer endgültigen Zellkonzentration von 5 x 10 5 Zellen / ml. Zwei verschiedene Bedingungen wurden verglichen: (1) Durchschnitt interstitielle flow = 0,1 &amp;mgr; s -1 und (2) statischen Zustand = keine messbare Durchfluss (Abbildung 1). Nach 24 Stunden wurden die Zellen, die durch die Poren der Membran eingedrungen mit DAPI gefärbt die Zellzählung erleichtern. 2 zeigt ein repräsentatives Bild der befallenen Zellen. Unter Hellfeld, sind nur die Poren der Membran sichtbar. Fluoreszenz-, die DAPI-gefärbten Nuklei wurden zum Zählen von Zellen und die Phalloidin gefärbt F-Aktin-Strukturen wurden verwendet, um die Zellkörper (optional) zu visualisieren. Mit Hilfe eines Student-t-Test unter Annahme gleicher Varianzen haben wir gezeigt, dass interstitielle Flow signifikant erhöht MDA-MB-435S Zellinvasion von 2,3-fache gegenüber der statischen Bedingungen (p = 0,003) (Abbildung 3). Diese corroborates zu ähnlichen Ergebnissen (aber mit unterschiedlichen Matrix-Bedingungen und sind daher Strömungsgeschwindigkeiten) mit dieser Zelllinie 13-14. <img alt="1" src="/files/ftp_upload/4159/4159fig1.jpg" /> 1. Schematische Darstellung der 3-D-interstitiellen Fluidströmung Invasionshemmtest. Bereiten Sie zuerst Gel-Lösung unter Verwendung von geeigneten Konzentrationen und Volumina. Dann fügen Sie Zellen auf Gel-Lösung und Transfer zum Zellkultur-Inserts. Schließlich fügen Sie entsprechende Volumen von Medien auf jede Bedingung und inkubieren. Interstitielle Flüssigkeit fließt durch einen Fluiddruck Kopf getrieben. <img alt="2" src="/files/ftp_upload/4159/4159fig2.jpg" /> 2. Transmigrierten MDA-MB-435S-Zellen auf der Membran. A) Bild von der Membran unter Hellfeld;; befallenen Zellen wurden nach unserer interstitiellen Flüssigkeit fließen Invasion Assay und mit DAPI gefärbt und Alexa Fluor 488-Phalloidin zu erleichtern Zählen der befallenen Zellen fixiert B) DAPI staINED Zellkerne (blau); C) Alexa Fluor 488-Phalloidin angefärbt F-Aktin (in grün). Maßstabsbalken repräsentiert 50 um. <img alt="Abbildung 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> Abbildung 3. Erhöhte Invasion von MDA-MB-435S-Zellen unter Strom. Zellinvasion nach 24 Stunden deutlich durch interstitielle flow (p = 0,003) verbessert. Die Ergebnisse sind der Durchschnitt statischen Zustand normalisiert und Werte repräsentieren den Mittelwert ± SEM von 6 Zellkultur-Inserts.

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Hier haben wir eine Methodik zur Quantifizierung der Wirkung der Strömung auf interstitielle Tumorzellinvasion, unter Verwendung von Zellen in einem 3-D-Matrix in einer Zellkultureinsatzes eingebetteten beschrieben. Diese und ähnliche Verfahren sind verwendet worden, um die Wirkung der interstitiellen Strömung auf einer Vielzahl von Zelltypen 13-15, 17 studierst. Unser Ansatz teilweise imitiert die Matrix Mikroumgebung des Tumors mit Kollagen Typ I und Matrigel, die Proteine ​​in der Basalmembran von E...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Collagen (Rattenschwanz) </td><td> BD </td><td> 354236 </td><td> Halten sterilen </td></tr><tr><td> Millicell Zellkultureinsatzes </td><td> Millipore </td><td> PI8P01250 </td><td> 8 mu m Porendurchmesser, Polycarbonat-Membran </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354234 </td><td> Halten sterilen </td></tr><tr><td> PBS </td><td> Sigma Aldrich </td><td> 100M-8202 </td><td> 10x für die Vorbereitung Gel-Lösung, 1x für Waschschritte </td></tr><tr><td> Natriumhydroxid, 1,0 N-Lösung </td><td> Sigma Aldrich </td><td> S2770 </td><td> Halten sterilen </td></tr><tr><td> DMEM 1X </td><td> CellGro </td><td> 10 bis 013-CV </td><td> Halten sterilen </td></tr><tr><td> Fötalem Rinderserum </td><td> Atlanta Biologicals </td><td> 511150 </td><td> Halten sterilen </td></tr><tr><td> Penicillin Streptomycin </td><td> CellGro </td><td> 30002CI </td><td> Halten sterilen </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> X100-500 ml </td><td> 0,5% in PBS </td></tr><tr><td> Paraformaldehyd </td><td> Fisher Scientific </td><td> 04042-500 </td><td> 4% in PBS </td></tr><tr><td> Deionisiertes Wasser </td><td></td><td></td><td> Halten sterilen </td></tr><tr><td> 4 &#39;,6-diaminido-2-phenylindol (DAPI) </td><td> MP Biomedicals </td><td> 0215757401 </td><td> 1 mg / ml Stammlösung </td></tr><tr><td> Montage-Lösung </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Trypsin-EDTA </td><td> CellGro </td><td> 25-052-CI </td><td> Halten sterilen </td></tr></tbody> &lt;/ Table&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

Das Wachstum und die Progression der meisten soliden Tumoren sind abhängig von der anfänglichen Transformation der Krebszellen und ihre Reaktion auf Stroma-assoziierten Signalmoleküle in der Tumor-Mikroumgebung 1. Bisher hat die Forschung auf die Tumor-Mikroumgebung in erster Linie auf die Tumor-Stroma-Interaktionen 2.1 konzentriert. Allerdings ist die Tumor-Mikroumgebung auch eine Vielzahl von biophysikalischen Kräfte, deren Wirkungen noch wenig verstanden. Diese Kräfte sind biomechanischen Folgen des Tumorwachstums, die zu Veränderungen in der Genexpression, Zellteilung, Differenzierung und Invasion 3 führen. Dichte-Matrix 4, Steifigkeit 5-6, 6-7 und Struktur, interstitielle Flüssigkeitsdruck 8, und der interstitiellen Flüssigkeit fließen 8 sind alle während der Tumorprogression verändert. Interstitielle Fluidströmung insbesondere eine höhere Tumoren im Vergleich zu normalem Gewebe 8-10. Die geschätzte interstitiellen Flüssigkeit flow Geschwindigkeiten wurden gemessen und in dem Bereich von 0,1-3 um s -1, je nach Größe des Tumors und Differenzierung 9, 11. Dies ist aufgrund des erhöhten interstitiellen Druckes mit Tumor-induzierte Angiogenese und erhöhte Gefäßpermeabilität 12 hervorgerufen wird. Interstitielle Fluidströmung hat sich gezeigt, um das Eindringen von Krebszellen 13-14, vaskulären Fibroblasten und glatten Muskelzellen 15 erhöht wird. Dieses Eindringen kann auf autologen chemotaktische Gradienten um Zellen in 3-D 16 erzeugt oder erhöht Matrix-Metalloproteinase (MMP)-Expression 15, Chemokin Sekretion und Expression Zelladhäsionsmolekül 17. Jedoch ist der Mechanismus, durch den Zellen zu erfassen Fluidströmung nicht gut verstanden. Zusätzlich zu verändern Tumorzelle Verhalten moduliert interstitiellen Flüssigkeit fließen die Aktivität anderer Zellen in der Tumor-Mikroumgebung. Es ist mit (a) Fahren Differenzierung von Fibroblasten in tumorpromovierenden myofibr verbundenoblasts 18, (b) Transportieren von Antigenen und anderen löslichen Faktoren in Lymphknoten 19, und (c) Modulieren lymphatischen Endothelzellen Morphogenese 20. Die hier vorgestellte Technik auferlegt interstitiellen Flüssigkeit fließen auf Zellen in vitro und quantifiziert deren Auswirkungen auf Invasion (Abbildung 1). Diese Methode wurde in mehreren Studien veröffentlicht worden, um die Auswirkungen der Strömung auf Stroma und die Invasion von Krebszellen 13-15, 17 zu messen. Durch Ändern der Matrix-Zusammensetzung, dem Zelltyp, und Zellkonzentration, kann dieses Verfahren auf andere Krankheiten und physiologische Systeme angewendet werden, um die Auswirkungen der interstitiellen Strömung auf zellulärer Prozesse, wie Invasion, Differenzierung, Proliferation und die Genexpression zu untersuchen.

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

Interstitielle Fluidstrom wird in soliden Tumoren erhöht und modulieren kann Tumorzellinvasion. Hier beschreiben wir eine Technik, um interstitielle Flüssigkeit fließen, um Zellen in einer Matrix eingebettet zu bringen und dann die seine Auswirkungen auf Zellinvasion. Diese Technik kann leicht angepasst werden, um andere Systeme zu studieren.

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 ...

three-dimensional-cell-culture-model-for-measuring-effects

Research

JoVE Journal

Bioengineering

16.4K Aufrufe.  Drexel University. Interstitielle Fluidstrom wird in soliden Tumoren erhöht und modulieren kann Tumorzellinvasion. Hier beschreiben wir eine Technik, um interstitielle Flüssigkeit fließen, um Zellen in einer Matrix eingebettet zu bringen und dann die seine Auswirkungen auf Zellinvasion. Diese Technik kann leicht angepasst werden, um andere Systeme zu studieren.

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Alginat Hydrogele für Three-Dimensional Organ Culture der Eierstöcke und Eileitern

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Forschung

Biotechnik

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Untersuchung der dreidimensionalen Strömungstrennung, die durch ein Modell Vocal Fold Polyp induziert wird

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

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

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

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 ...

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 ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gentransfektion in Richtung Spheroid Zellen auf mikrostrukturierten Kulturplatten für genetisch veränderte Zelltransplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Die Herstellung von Inverted Kolloid-Kristallen Poly (Ethylenglycol) Scaffold: Eine dreidimensionale Zellkultur-Plattform für Leber-Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Zellwanderung in dreidimensionales In Vitro Charakterisierung Wunde Umgebungen

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Bewertung der Keratinozyten Verbreitung auf zwei- und dreidimensionale Typ I Kollagen Substrate

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Verwendung von mehrschichtigem Hydrogel Bioink im dreidimensionalen Bioprinting für homogene Zellverteilung

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Primärklärung der CHO geernteten Zellkulturflüssigkeit mittels eines akustischen Separators

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...