Finanzierung für diese Arbeit wurde durch die American Heart Association (16SDG29870005) und der North Carolina State University Research und Innovation Seed-Finanzierung zur Verfügung gestellt.

1. Tag1: Zelle und Reagenz Vorbereitung Hinweis: alle Zell- und Reagenz Vorbereitung sollte in einem biologischen Sicherheitsschrank gegen Kontamination der Proben durchgeführt werden.
<ol><li>Warm gefiltert Dulbecco &#39; s Modified Eagle &#39; s Medium (DMEM), fetale bovine Serum (FBS), L-Glutamin und Penicillin/Streptomycin in einem 37 ° C Wasserbad.</li>
	<li>Vorbereiten Neugeborenen menschlichen dermalen Fibroblasten (HDFn) Medien durch Ergänzung erwärmt DMEM mit 10 % FBS, 1 % Penicillin/Streptomycin und 1 % L-Glutamin.</li>
	<li>Tauwetter ein Fläschchen mit gefrorenen HDFns und Zentrifuge für 8 min. bei 200 X g Kryokonservierung Medium zu entfernen. Zellen in vorbereitete HDFn Medien bei einer Dichte von 1 x 10 6 Zellen/mL und Samen in einer T-75 Kultur Flasche aufzuwirbeln.</li>
	<li>Ort Kolben in einem Inkubator (37 ° C, 5 % CO 2) erlauben Zellproliferation.</li>
	<li>Store HDFn Media bei 4 ° c</li>
	<li>Pro und anti migratory Reagenzien vorzubereiten, wie gebraucht.</li>
</ol> 2. 3. Tag: Sphäroid Vorbereitung <ol><li>führen Sie die folgenden in eine Kultur-Haube, Kontamination der Proben zu verhindern.</li>
	<li>Wenn Zellen erreichen 80 % Konfluenz aspirieren Sie Medien und Hinzufügen von 0,25 % 5 mL Trypsin-EDTA. Shop-Kolben in einem Inkubator 37 ° C für 5 min, Zellen von der Oberfläche der Küvette vollständig zu lösen.</li>
	<li>Fügen Sie 5 mL erwärmten Medien in den Kolben, die Trypsin neutralisieren.</li>
	<li>In einem Microcentrifuge Schlauch mischen, 10 μL Trypan blau und 10 μl Zellsuspension.</li>
	<li>Zentrifuge der ursprünglichen Zellsuspension für 8 min bei 200 X g.</li>
	<li>Während die Zellsuspension zentrifugiert wird, ist ein Hemocytometer 10 μl Zellsuspension Trypan Blau hinzu, und führen Sie eine Zellzahl.</li>
	<li>Nach Zentrifugation, Aspirieren Medien ohne verdrängen die Zelle Pellet. Aufschwemmen der Zelle Pellet in einer Konzentration von 1,25 x 10 5 Zellen/mL.</li>
	<li>Fügen Sie 5 mL sterilem Phosphat gepufferte Kochsalzlösung (PBS) auf den Boden einer 10 cm Petrischale Erhalt eine hydratisierte Umgebung während des Wachstums der Sphäroid.</li>
	<li>Drehen Sie den Deckel der Petrischale. Fügen Sie mehrere 20 μL Tropfen der Zellsuspension auf der inneren Oberfläche des Deckels Petrischale.</li>
	<li>Schnell den Deckel wieder umkehren und legen Sie sie wieder auf den Boden der Schale, ohne dabei die hängenden Tropfen zu verdrängen. Abbildung 1 zeigt erfolgreich gebildete hängenden Tropfen.</li>
	<li>Vorsichtig legen Sie die Schale in einem 37 ° C Inkubator und lassen Sphäroide wachsen in einer hängenden Tropfen Kultur für einen Zeitraum von 72 h.</li>
</ol> 3. 6. Tag: Einbettung Sphäroide in 3D Matrix <ol><li>führen Sie die folgenden in eine Kultur-Haube, Kontamination der Proben zu verhindern.</li>
	<li>Für die Bildung der ersten Schicht Fibrin, erstellen eine Gerinnsel Lösung ausreichend Volumen hinzufügen 100 μL Gerinnsel Lösung pro Bohrloch für so viele Brunnen wie erforderlich (1 Sphäroid pro Well). Klumpen bestehen aus 10 % 10 X HEPES Puffer nach Volumen (250 mM HEPES, 1500 mM NaCl, 50 mM CaCl 2, pH 7,4), 2 mg/mL menschlichen Fibrinogen und 0,1 U/mL menschliches alpha Thrombin. Der Rest des Bandes Gerinnsel besteht aus hochreinem Wasser.
	<ol><li>Add Thrombin zuletzt und schnell hinzufügen Lösung zur gewünschten Brunnen in einem 48-Well-Platte um Klumpen zu vermeiden dadurch vor dem Brunnen hinzugefügt wird.</li>
	</ol></li> <li>Sanft schütteln/Tap-well-Platte um sicherzustellen, dass Fibrin Gerinnsel Mischung auch die gesamte Beschichtung ist, und platzieren Sie well-Platte in den Inkubator für 1 h Gerinnsel Unterschicht zu polymerisieren zu ermöglichen. Schütteln Sie well-Platte hart genug, um Blasenbildung zu induzieren nicht; einfach Felsplatte als nötig, bis die Gesamtheit des Brunnens beschichtet ist. Wenn größere Gerinnsel Mengen verwendet werden, kann dieser Schritt nicht notwendig sein.</li>
	<li>Sphäroide auf die Fibrin-Schicht übertragen.
	<ol><li>Entfernen der Brunnen Platte und die Petrischale mit Sphäroide in den hängenden Tropfen Kultur aus dem Inkubator und Sphäroide unter dem Mikroskop zu untersuchen. Legen Sie die Schale in der Kultur-Motorhaube.</li>
		<li>Invertieren vorsichtig den Deckel von der Petrischale für den Zugriff auf die hängenden Tropfen Kulturen.</li>
		<li>Mit einer Nadel 21 G eine 1 mL Spritze sorgfältig übertragen ein Tropfen aus dem umgekehrten Deckel in der Mitte des jeweils gut kümmert sich nicht um die Beschichtung der Unterseite des Brunnens polymerisierten Fibrin-Schicht zu durchbohren. Um den Tropfen effektiv zu übertragen, ziehen Sie die Tropfen in die Nadel. Ziehen den Kolben zurück, sobald der gesamte Rückgang in der Nadel ist zu stoppen; die Tropfen zu weit zurückziehen kann einführen Luftblasen, die effektive Sphäroid Transfer oder nachfolgenden Bildgebung stören können.</li>
		<li>Untersuchen die well-Platte unter dem Mikroskop um sicherzustellen, dass Sphäroide ordnungsgemäß in allen gewünschten Vertiefungen übertragen haben.</li>
	</ol></li> <li>Für die Bildung der zweiten Schicht von Fibrin, erstellen eine andere Gerinnsel Lösung verwenden die gleiche Vorbereitung wie oben für die Erstellung der ersten Fibrin-Schicht (Schritt 3.2). Sorgfältig pipette 100 μL auf jedem Sphäroid-haltigen Tropfen und untersucht unter dem Mikroskop wieder, um sicherzustellen, dass die Sphäroide sind noch intakt und nicht an den Rändern der Brunnen gerückt.</li>
	<li>Rückkehr der well-Platte in den Inkubator und die zweite Schicht für 1 h polymerisieren zu ermöglichen</li>
</ol> 4. 6. Tag: Zugabe von Pro-wandernde oder hemmende Faktoren <ol><li>Warm HDFn Medien auf 37 ° c</li>
	<li>Führen Sie die folgenden Schritte in eine Haube um Kontaminationen zu vermeiden.</li>
	<li>Bereiten Sie Medien für jede Gruppe, einschließlich der entsprechende Konzentrationen von Pro/anti-migratory Agenten zu evaluierenden.</li>
	<li>Entfernen der well-Platte aus Inkubator und in eine Kultur Kapuze.</li>
	<li>Hinzufügen 500 μL der Standardmedien in jede Vertiefung der Kontrollgruppe Sphäroid, 500 μL der hemmenden Medien in jede Vertiefung des Arbeitskreises Migration gehemmt Sphäroid und 500 μl Pro wandernden Medien in jede Vertiefung des Arbeitskreises Migration-enhanced Sphäroid.</li>
	<li>Rückkehr der well-Platte in den Inkubator.</li>
	<li>Änderung Medien alle 48 h.</li>
</ol> 5. 6-9 Tage: Evaluation of Cell Migration und Proliferation <ol><li>Bild Sphäroide mit Hellfeld Mikroskopieren sofort nach Zugabe von Medien (0 h) und dann alle 24 h für 72 h Optional: zu jedem Zeitpunkt eine Z-Stapel-Bild nehmen, um festzustellen, ob Migration ist in Ebenen außerhalb des primären Fokus auftreten. Erfahrungsgemäß tritt dies nicht auf; Es ist jedoch nützlich, um sicherzustellen, dies ist der Fall bei jedem Versuch.</li>
	<li>Einsatz ImageJ Zelle Auswuchs in jede Vertiefung durch Analyse Prozent Zuwachs im Bereich für jedes Sphäroid durch Rückverfolgung der Zellenrahmen für jedes Sphäroid an jedem Punkt Mal in Folge und mit bestimmen die &#34; Maßnahme &#34; Funktion, um die Fläche zu berechnen.</li>
</ol>

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Dieses Protokoll ermöglicht die Prüfung der Zellwanderung in der Wundheilung ECMs über eine in-vitro- 3D Modell zugeordnet. Ein entscheidender Schritt für die ordnungsgemäße Durchführung des Verfahrens ist die richtige Entwicklung der Fibroblasten Sphäroide; Zellkonzentration während der Vorbereitung wurde optimiert, um eine Ausgangskonzentration von 2.500 Zellen/Tropfen, geben so dass Sphäroide zu bilden zuverlässig über eine Inkubationszeit von 72 h. Wenn eine unzureichende Anzahl von Zellen verwen...

Wundheilung ist ein komplexer Prozess, der die Wiederherstellung der Gewebe Integrität nach Verletzung führt. Dieser Prozess wird in vier überlappende Phasen Blutstillung, Entzündungen, Verbreitung und Umbau, umgeben aufgeteilt die jeweils geregelt sind durch eine komplexe Kombination von löslichen Queues, Zelle-Matrix Interaktionen und Zell-Zell-Kommunikation. 1 , 2 die Orchestrierung dieser Signale steuert eine Vielzahl von zellulären Reaktionen bei der Wundheilung einschließlich allem Zellmigration. 3 , 4 Zellwanderung ist ein sehr dynamischer Prozess abhängig von zellulären Phänotypen und die biochemischen und biophysikalischen Eigenschaften des extrazellulären Matrix Milieus. 5 Zellen Sonde kontinuierlich ihre extrazellulären Matrix Umwelt durch Integrin-vermittelte Wege; Dieser Prozess ermöglicht Zellen, eine Vielzahl von Matrixeigenschaften einschließlich der Topographie, der Steifigkeit und der Entbindung zu spüren. Zweidimensionale (2D) Analyse der Zellwanderung zeigt, dass die Migration von Lamellipodia Bildung an der Vorderkante durch die Generierung von Aktin-Filament-Bundles angetrieben wird. Nachträgliche Bildung von fokalen Adhäsionen an der Vorderkante, in Kombination mit Actomyosin getrieben Kontraktion ein- und Ausfahren an der Hinterkante treibt die Zelle. 6 dreidimensionale (3D) zelluläre Migration ist derzeit nicht bekannt, jedoch neuere Studien zeigen, dass 3D Migration deutlich anders als die zuvor Mechanismus in 2D beschriebenen und wird wahrscheinlich von alterative Mechanismen gesteuert. Beispiel: neuere Studien von Petrie Et Al. gezeigt, dass je nach den Eigenschaften des ECM, Zellen in 3D Matrizen zwischen Lamellipodium und Lobopodium getrieben Mechanismen der Migration wechseln können. 7 da Zellwanderung ein wesentlicher Bestandteil der Wundheilung Reaktion ist, 3D-Modelle von Zellwanderung sind entscheidend für das Verständnis der physiologischer Reaktionen auf verschiedene Reize bei der Wundheilung. Obwohl Zelle Wanderungsverhalten auf 2D Oberflächen stark variieren von Migration in 3D Matrizen nachgewiesen wurde, nutzen die aktuelle in-vitro- Modelle der Zellwanderung während der Wundheilung, einschließlich der etablierten Scratch Test 2D Monolayer-Kulturen. 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 vor kurzem entwickelt Tests eine 3D Matrix verwendet haben, aber noch Kulturzellen in einer Monolage auf der Matrix begrenzt somit die Fähigkeit, wirklich die Dreidimensionalität der Zelle Umwelt rekapitulieren in-vivo. 15
Die hier beschriebene Methode soll Zellwanderung in einem physiologisch relevanten 3D-Modell, anstatt auf eine 2D Oberfläche zu studieren, um ein robuster Verständnis von Migration Antworten verbunden sind mit den angewandten reizen. Diese Methode ermöglicht die Bewertung der Zellwanderung innerhalb einer 3D Fibrin-Gerinnsel-Matrix im Beisein von Faktoren, die zu aktivieren oder hemmen Wege Zellwanderung zugeordnet. Für diese Methode durch die kritische Rolle der Fibrin in den frühen Phasen der Wundheilung wurde eine Fibrin-Matrix gewählt; Folgende Verletzungen, ein Fibrin-Gerinnsel entsteht innerhalb der Umgebung der Wunde Blutverlust einzudämmen und dient als Gerüst für die erste Zelle eindringen während der Reparatur von Gewebe und Umbau. 2 , 16 , 17 , 18 , 19 , Darüber hinaus 20 Fibrin wird klinisch als eine chirurgische Dichtungsmittel verwendet und die Zusammensetzung der Dichtstoffe Zellwanderung und ultimative Heilung Ergebnisse gebunden worden ist. Einer früheren Studie von Cox Et Al. Migration von Fibroblasten mit Fibrin Gerinnsel bestehend aus unterschiedlichen Fibrin und Thrombin Konzentrationen zum Zwecke der Optimierung ein Fibrin Dichtungsmittel untersucht. In diesen Studien wurde die Wanderung von Zellen aus Fibrin Gerinnsel auf Plastikschalen quantifiziert. 20 hier präsentieren wir eine Methode, die zur Quantifizierung der Migration von Zellen innerhalb der 3D Fibrin-Umgebung ermöglicht. Während die Methode, die speziell in diesem Protokoll beschriebenen Fibrin verwendet, kann dieses Modell leicht geändert werden, um alternative Matrixmaterialien zu verwenden, wie gewünscht, z. B. Kollagen oder andere 3D Matrizen. Darüber hinaus präsentieren wir die Verwendung von Fibroblasten Sphäroide in diesem Assay, weil Fibroblasten Migration in das Wundbett für Synthese der extrazellulären Matrix und Gewebe Reparatur/Umbau von höchster Bedeutung ist. Jedoch während der Reparatur sind Prozess Neutrophile die erste Zelle Art in das Wundbett, gefolgt von Makrophagen wandern. Fibroblasten beginnen dann die Wundumgebung ca. 48 Stunden nach der ersten Verletzung zu Beginn der Proliferativen Phase der Wundheilung zu infiltrieren. 19 dieser Assay kann leicht geändert werden, um zählen Neutrophile, Makrophagen oder anderen Zelltypen zu beurteilen, wie Änderungen in Fibrin Gerinnsel Struktur Migration dieser Zelltypen beeinflussen. 21
Um diesen Test zu implementieren, wird zunächst ein Sphäroid Fibroblasten gebildet eine Modifikation der zuvor beschriebenen für Stammzelle-Kultur Techniken. 22 Fibroblasten Sphäroid wird anschließend in einer 3D Fibrin-Matrix übertragen, und Auswuchs kann dann leicht überwacht und über mehrere Tage quantifiziert. Dieses Protokoll berücksichtigt die 3D physiologische Systeme durch das Einbetten von 3D Zelle Sphäroide innerhalb einer Fibrin-Matrix, wodurch die Einschränkungen in Relevanz durch eine mit einer 2D Wachstumsoberfläche eine Zelle Monolage auf Modell Wanderungsverhalten herbeigeführt, zwar noch ermöglicht zur Auswertung der Zelle Verhalten in einer streng kontrollierte in-vitro- Umgebung.

<table border="0" cellpadding="0" cellspacing="0" width="692">
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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Materials</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Dulbecco&#39;s Modified Eagle&#39;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td>
			<td style="width:91px;">VWR</td>
			<td>VWRL0148-0500</td>
			<td style="width:217px;">DMEM already containing L-glutamine can also be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td>
			<td style="width:91px;">VWR</td>
			<td>10861-594</td>
			<td style="width:217px;">Dishes of any size can be used for hanging drop culture&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>12306C-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">L-glutamine</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td>ICN1680149</td>
			<td style="width:217px;">Not needed if using DMEM that already contains L-glutamine&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Human Dermal Fibroblasts, neonatal</td>
			<td style="width:91px;">Thermo Fisher</td>
			<td>C0045C</td>
			<td style="width:217px;">Can be replaced with other cell type of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">21 G x 1 1/2&#39; needle</td>
			<td style="width:91px;">BD&#160;</td>
			<td>305167</td>
			<td>22 G x 1 1/2 needles will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1 mL syringe</td>
			<td style="width:91px;">VWR</td>
			<td>89174-491</td>
			<td>Syringes of any volume can be used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td>
			<td style="width:91px;">VWR</td>
			<td>82051-004&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>FIB 3</td>
			<td style="width:217px;">Can be replaced with matrix protein of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Alpha Thrombin</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>HT 1002a</td>
			<td style="width:217px;">May not be necessary depending on matrix protein of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Equipment</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSL2 cell culture hood</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell culture incubator</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Inverted microscope with 10X objective</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Centrifuge compatibile with 15 mL tubes</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing cell migration within three-dimensional in vitro wound environments

Derzeit die meisten in-vitro- Modelle der Wundheilung, wie etablierte Scratch-Assays, Studium Zelle Migration und Wunde Schließung auf zweidimensionalen Flächen beinhalten. Allerdings ist die physiologische Umgebung welche in Vivo Wundheilung erfolgt dreidimensional als zweidimensionale. Es wird immer deutlicher, dass Zelle Verhalten unterscheidet sich stark in zweidimensionalen vs. dreidimensionale Umgebungen; Daher gibt es eine Notwendigkeit an mehr physiologisch relevanten in-vitro- Modelle für das Studium Zelle Migration Verhaltensweisen in Wundverschluss. Die hier beschriebene Methode ermöglicht das Studium der Zellwanderung in einem dreidimensionalen Modell, das physiologische Bedingungen besser als vorher festgelegten zweidimensionale Scratch Assays widerspiegelt. Dieses Modell soll Zelle Auswuchs über die Prüfung der Zellmigration Weg ein Sphäroid Körper eingebettet innerhalb einer Fibrin-Matrix im Beisein von Pro oder anti migratory Faktoren zu bewerten. Mit dieser Methode Zelle Auswuchs aus dem Sphäroid Körper in einer dreidimensionalen Matrix beobachtet werden und im Laufe der Zeit durch Brightfield Mikroskopie und Analyse von Sphäroid Körperbereich leicht quantifizierbar ist. Die Wirkung von Pro-wandernde oder hemmende Faktoren auf Zellwanderung kann auch in diesem System ausgewertet werden. Diese Methode bietet eine einfache Methode des Analysierens Zellwanderung in dreidimensionale Wunde verbunden Matrizen in-vitro-Forscher, wodurch sich die Relevanz der in-vitro- Studien Zelle vor der Verwendung von in-Vivo -Tier Modelle.

Sphäroid Kultur kann genutzt werden, um die Wirkung der pro und Anti-wandernde Agenten auf Fibroblasten Migration in-vitro-erfolgreich bewerten 3D Fibroblasten Sphäroide können gebildet werden, über hängende Tropfen Kultur über einen Zeitraum von 72 h (Abbildung 1). Im Anschluss an die Kulturdauer Sphäroide innerhalb der Gerinnsel-Matrix eingebettet sind und abgebildet mit Brightfield Mikroskopie (Abbildung 2</s...

Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Das Ziel dieses Protokolls ist es, die Wirkung der Pro und anti migratory Faktoren auf Zellwanderung innerhalb einer dreidimensionalen Fibrin-Matrix bewerten.

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 ...

characterizing-cell-migration-within-three-dimensional-vitro-wound

Research

JoVE Journal

Bioengineering

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

7.6K Aufrufe.  North Carolina State University and The University of North Carolina - Chapel Hill. Das Ziel dieses Protokolls ist es, die Wirkung der Pro und anti migratory Faktoren auf Zellwanderung innerhalb einer dreidimensionalen Fibrin-Matrix bewerten.

Serie: Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Dreidimensionale Optische Auflösung photoakustische Mikroskopie

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Forschung

Biotechnik

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Electric Field-gesteuerte gerichtete Wanderung von neuralen Vorläuferzellen in 2D-und 3D-Umgebungen

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Mikroinjektion Wound Assay und<em&gt; In-vivo-</em&gt; Lokalisierung von Epidermal Wundreaktion Reporter in<em&gt; Drosophila</em&gt; Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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

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

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

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

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

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

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Herstellung und Analyse von<em&gt; In Vitro</em&gt; Three Dimensional Mammakarzinoms Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Technik Dreidimensionale Epithelgewebe Eingebettet in extrazellulären Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Kultivierung von Säugerzellen in dreidimensionale Peptid Gerüste

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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. ...