El financiamiento de este trabajo fue proporcionado a través de la Asociación Americana del corazón (16SDG29870005) y la investigación de la Universidad del estado de Carolina del norte y financiación innovación.

1. día 1: la célula y preparación de los reactivos Nota: toda la preparación de la célula y el reactivo debe realizarse en un gabinete para prevenir la contaminación de las muestras de seguridad biológica.
<ol><li>Caliente filtrada Dulbecco &#39; s Modified Eagle &#39; s medio (DMEM), suero bovino fetal (FBS), L-glutamina y penicilina/estreptomicina en un baño de agua de 37 ° C.</li>
	<li>Prepare medios de fibroblastos dérmicos humanos neonatales (HDFn) complementando calentado DMEM con 10% FBS, penicilina/estreptomicina de 1% y 1% L-glutamina.</li>
	<li>Descongelar un vial de congelados HDFns y centrifugue durante 8 min a 200 x g para eliminar medio criopreservación. Resuspender las células en medios de HDFn preparados a una densidad de 1 x 10 6 células/mL y semilla en un frasco de cultura T-75.</li>
	<li>Matraz de lugar en una incubadora (37 ° C, 5% CO 2) para permitir la proliferación celular.</li>
	<li>Tienda HDFn media a 4 ° C.</li>
	<li>Preparar los pro y anti migratory reactivos según sea necesario.</li>
</ol> 2. Día 3: Esferoide preparación <ol><li>haga lo siguiente en una campana para prevenir la contaminación de las muestras de cultura.</li>
	<li>Cuando las células alcanzan el 80% de confluencia, aspirar los medios de comunicación y añadir 5 mL de 0.25% tripsina-EDTA. Frasco de tienda en una incubadora de 37 ° C por 5 min separar completamente las células de la superficie del frasco.</li>
	<li>Añadir 5 mL de medio caliente al matraz para neutralizar la tripsina.</li>
	<li>En un tubo de microcentrífuga, mezclar 10 μL de azul tripán y 10 μL de la suspensión celular.</li>
	<li>Centrifugar la suspensión de la célula original durante 8 min a 200 x g.</li>
	<li>Mientras que la suspensión de células es ser centrifugada, añadir 10 μL de la suspensión de células de azul de tripano a un hemocitómetro y realizar un conteo de.</li>
	<li>Después de centrifugación, aspirar los medios de comunicación sin el desalojamiento de la pelotilla de la célula. Resuspender el precipitado de células en una concentración de 1,25 x 10 5 células/mL.</li>
	<li>Añadir 5 mL de tampón de fosfato estéril salino (PBS) en la parte inferior de una placa de Petri para mantener un ambiente hidratado durante el crecimiento del esferoide de 10 cm.</li>
	<li>Invertir la tapa de la caja Petri. Añadir varias 20 gotas de μL de la suspensión de células en la superficie interna de la tapa de la caja de Petri.</li>
	<li>Rápidamente invertir la tapa nuevamente y colocar en el fondo de la fuente, teniendo cuidado de no desplazar las gotas de la suspensión. la figura 1 ilustra correctamente formados colgante gotas.</li>
	<li>Cuidadosamente coloque el plato en una incubadora de 37 ° C y esferoides en colgante gota cultura durante un período de 72 h.</li>
</ol> 3. Día 6: Incorporación de esferoides en matriz 3D <ol><li>haga lo siguiente en una campana para prevenir la contaminación de las muestras de cultura.</li>
	<li>Para la formación de la primera capa de fibrina, crear una solución de coágulo de volumen suficiente para añadir 100 μL de solución de coágulo por pozo para tantos pozos como necesaria (1 esferoide por pozo). Coágulos se componen de 10% 10 X HEPES del almacenador intermediario por volumen (250 mM HEPES, 1500 mM NaCl, 50 mM CaCl 2, pH 7,4), fibrinógeno humano 2 mg/mL y 0,1 U/mL humano alfa de la trombina. El resto del volumen del coágulo se compone de agua ultrapura.
	<ol><li>Añadir trombina última y rápidamente agregue una solución a pozos deseadas en una placa de 48 pozos para impedir que coágulos de polimerizarse antes de ser añadido a los pozos.</li>
	</ol></li> <li>Agitar suavemente/tap bien placa para asegurar que la mezcla del coágulo de fibrina es capa todo el bien y luego colocar la placa bien en la incubadora durante 1 hora permitir que la capa inferior de clot polimerizar. No sacuda la placa bien dura inducir la formación de burbujas; simplemente la placa de roca como sea necesario hasta que la totalidad del pozo es revestida. Si se utilizan volúmenes de coágulos más grandes, este paso puede no ser necesario.</li>
	<li>Transferencia esferoides a la capa de fibrina.
	<ol><li>Quitar bien la placa y la placa de Petri que contiene esferoides en el colgante gota de cultivo de la incubadora y examinan esferoides bajo un microscopio. Luego coloque el plato en la campana de cultura.</li>
		<li>Invertir cuidadosamente la tapa de la caja Petri para permitir el acceso al colgante gota culturas.</li>
		<li>Cuidadosamente con una aguja de 21 G conectada a una jeringa de 1 mL, transferir una gota de la tapa invertida en el centro de cada bien, teniendo cuidado de no para perforar la capa de fibrina polimerizada cubriendo el fondo del pozo. Para transferir efectivamente la gota, Jale lentamente la caída en la aguja. Dejar de tirar el émbolo hacia atrás tan pronto como la caída de toda está dentro de la aguja; tirando la caída hacia atrás demasiado lejos puede introducir burbujas de aire, que pueden alterar imágenes posteriores o transferencia efectiva esferoide.</li>
		<li>Examinar la placa bien bajo un microscopio para asegurarse de que esferoides han transferido correctamente en pocillos todo deseados.</li>
	</ol></li> <li>Para la formación de la segunda capa de fibrina, crear otra solución de coágulo, con la misma preparación que el anterior para la creación de la primera capa de fibrina (paso 3.2). Cuidadosamente Pipetee 100 μL en cada gota que contiene el esferoide y examinar bajo el microscopio para asegurarse de que los esferoides están todavía intactos y no han empujado a los bordes de los pozos.</li>
	<li>Devolver la placa bien a la incubadora y permitir que la segunda capa polimerizar por 1 h.</li>
</ol> 4. Día 6: Adición de factores inhibitorios o Pro migratorias <ol><li>medios de HDFn caliente a 37 ° C.</li>
	<li>Siga los siguientes pasos en una campana para evitar la contaminación.</li>
	<li>Preparar los medios de comunicación para cada grupo incluyendo las concentraciones adecuadas de agentes pro/anti-migratory evaluarse.</li>
	<li>Quitar la placa de la pozo de incubadora y el lugar en una campana de cultura.</li>
	<li>Añadir 500 μL de medio estándar a cada pocillo del grupo control esferoide, 500 μL de inhibitoria media a cada pocillo del grupo inhibe migración esferoide y 500 μL de medios pro migratorias en cada pocillo del grupo de mayor migración esferoide.</li>
	<li>Devolver la placa de la pozo a la incubadora de.</li>
	<li>Medios de cambio cada 48 h.</li>
</ol> 5. Días 6-9: evaluación de la migración celular y la proliferación <ol><li>imagen esferoides mediante microscopía brightfield inmediatamente después de la adición de medios (0 h) y luego cada 24 h por 72 h. opcional: en cada momento, tomar una imagen de z-stack para determinar si migración es que ocurre en planos fuera del área principal de enfoque. En nuestra experiencia esto no ocurre; sin embargo, es útil para asegurarse de esto es el caso en todos los experimentos.</li>
	<li>Uso de ImageJ para determinar extensión de células en cada pozo analizando por ciento aumento de superficie para cada esferoide trazar la frontera de la célula para cada esferoide en cada momento sucesivo y utilizando la &#34; medida &#34; función para obtener el área de.</li>
</ol>

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Los autores no tienen nada que revelar.

Este protocolo permite el examen de la migración celular en la cicatrización de heridas asociadas ECMs a través de un modelo 3D en vitro . Un paso crucial en la correcta ejecución del procedimiento es el correcto desarrollo de esferoides de fibroblastos; concentración celular durante la preparación se optimizó para dar una concentración inicial de 2.500 células/drop, permitiendo esferoides de forma fiable durante un período de incubación de 72 h. Si se usa un número insuficiente de células esferoide...

La cicatrización es un proceso complejo que resulta en la restauración de la integridad del tejido después de lesión. Este proceso se divide en cuatro etapas superpuestas de hemostasia, inflamación, proliferación y remodelación, que son cada uno regulado por una compleja combinación de señales solubles, las interacciones célula-matriz y célula-célula comunicaciones. 1 , 2 la orquestación de estas señales controla multitud de respuestas celulares en la cicatrización de heridas incluyendo, importantemente, migración de la célula. 3 , 4 migración celular es un proceso dinámico dependiente de fenotipos celulares y las propiedades bioquímicas y biofísicas de la medio de la matriz extracelular. 5 células sonda continuamente su entorno matriz extracelular a través de vías mediadas por integrinas; Este proceso permite a las células detectar una amplia gama de propiedades de la matriz incluyendo topografía, rigidez y aislamiento. Dos dimensiones (2D) el análisis de migración de la célula demuestra que la migración es impulsada por lamellipodia formación a la vanguardia a través de la generación de haces de filamentos de actina. Posterior formación de adherencias focales a la vanguardia, en combinación con actomyosin por contracción y retracción en el borde de fuga, unidades de la célula hacia adelante. 6 migración celular tridimensional (3D) en la actualidad no se entiende bien, sin embargo, estudios recientes demuestran que la migración 3D es marcadamente diferente a la anteriormente descrito mecanismo en 2D y es probablemente por mecanismos alterativo. Por ejemplo, estudios recientes por Petrie et al. demostró que, dependiendo de las propiedades de la ECM, las células matrices 3D pueden cambiar entre lamellipodium y lobopodium por mecanismos de la migración. 7 debido a migración de la célula es un componente crítico de la respuesta de cicatrización, modelos 3D de migración de la célula son críticos para la comprensión de respuestas fisiológicas a diferentes estímulos durante la cicatrización de heridas. Aunque el comportamiento migratorio de la célula en las superficies de la 2D se ha demostrado que varían mucho de la migración en matrices 3D, modelos en vitro más actuales de la migración celular durante el proceso, incluyendo el ensayo de rayado bien establecido, de cicatrización utilizan 2D cultivos de monocapa. 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 más recientemente desarrollado análisis han utilizado una matriz 3D, pero aún las células en una monocapa sobre la matriz, lo que limita la capacidad de realmente recapitular la tridimensionalidad del entorno de la célula de la cultura en vivo. 15
El propósito del método descrito en este documento es estudiar la migración de células en un modelo 3D fisiológicamente relevante, en lugar de en una superficie 2D, para tener una comprensión más robusta de las respuestas de migración asociados con los estímulos aplicados. Este método permite la evaluación de la migración de la célula dentro de una matriz del coágulo de fibrina 3D en presencia de factores que activan o inhiben vías asociadas con la migración de la célula. Una matriz de fibrina fue elegida para este método debido al papel crítico que desempeña fibrina en las primeras etapas de la cicatrización de heridas; siguientes lesiones, un coágulo de fibrina se formaron dentro de los entornos de la herida para detener la pérdida de sangre y sirven como un andamio para la infiltración de la célula inicial durante la reparación del tejido y remodelación. 2 , 16 , 17 , 18 , 19 , 20 además, fibrina se utiliza clínicamente como un sellante quirúrgico y la composición de los selladores ha estado ligada a la migración de la célula y el último los resultados de curación. Un estudio previo por Cox et al. investigado la migración de fibroblastos con coágulos de fibrina compuestos de fibrina variable y concentraciones de trombina con el fin de optimizar un sellante de fibrina. En estos estudios, se cuantificó la migración de células de coágulos de fibrina en platos de plástico. 20 aquí presentamos un método que permite la cuantificación de la migración de las células en el entorno 3D de la fibrina. Aunque el método descrito en el presente Protocolo específicamente utiliza fibrina, este modelo puede ser modificado fácilmente para utilizar materiales alternativos de matriz como se desee, como el colágeno u otras matrices 3D. Además, presentamos el uso de esferoides de fibroblastos en este ensayo debido a la migración de fibroblastos en el lecho de la herida es de suma importancia para la síntesis de matriz extracelular y tejido reparación/remodelación. Sin embargo, durante la reparación proceso neutrófilos son el primer tipo de células a migrar en el lecho de la herida, seguido por los macrófagos. Fibroblastos comienzan entonces a infiltrarse en el entorno de la herida aproximadamente 48 horas después de lesión inicial, al comienzo de la fase proliferativa del proceso de cicatrización. 19 este ensayo podría ser fácilmente modificado para incluir neutrófilos, macrófagos y otros tipos de células para evaluar cómo alteraciones en la estructura del coágulo de fibrina afectan la migración de estos tipos celulares. 21
Para aplicar este análisis, en primer lugar, un esferoide del fibroblasto se forma utilizando una modificación de las técnicas descritas para el cultivo de células madre. 22 el esferoide del fibroblasto se transfiere posteriormente en una matriz de fibrina 3D, y consecuencia puede entonces fácilmente supervisar y cuantificar durante varios días. Este protocolo tiene en cuenta el 3D de sistemas fisiológicos incrustando esferoides celulares 3D dentro de una matriz de fibrina, evitando así las limitaciones en relevancia provocada por usar una superficie de crecimiento 2D con una monocapa de células al comportamiento migratorio de la modelo, mientras que que permite para la evaluación del comportamiento de la célula en un ambiente altamente controlado en vitro .

<table border="0" cellpadding="0" cellspacing="0" width="692">
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		<col />
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		<col />
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	</colgroup>
	<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

Actualmente, mayoría en vitro de los modelos de cicatrización, como ensayos de rayado bien establecidos, implican estudiar celular migración herida cierre en superficies bidimensionales. Sin embargo, el ambiente fisiológico en que en vivo de cicatrización se lleva a cabo es tridimensional en lugar de dos dimensiones. Es cada vez más claro que el comportamiento celular difiere enormemente en dos dimensiones vs entornos tridimensionales; por lo tanto, hay una necesidad de más fisiológico relevantes en vitro modelos para estudiar comportamientos de migración celular en el encierro de la herida. El método aquí descrito permite el estudio de la migración celular en un modelo tridimensional que mejor refleja las condiciones fisiológicas que ensayos cero dos dimensiones previamente establecidos. El propósito de este modelo es evaluar consecuencia de células mediante el examen de la migración de la célula de un cuerpo esferoide incrustado dentro de una matriz de fibrina en presencia de factores de pro o anti migratory. Usando este método, fruto de célula del cuerpo esferoide en una matriz tridimensional puede ser observado y es fácilmente cuantificable en el tiempo mediante microscopía brightfield y análisis de la zona del cuerpo esferoide. El efecto de factores pro-migratorias o inhibitorios sobre migración de la célula puede también ser evaluado en este sistema. Este método ofrece a los investigadores con un método sencillo de análisis de migración de la célula en matrices tridimensionales herida asociada en vitro, aumentando la relevancia de in vitro células estudios previo a la utilización de animales en vivo modelos.

Cultura de esferoide puede ser utilizada para evaluar con éxito el efecto de agentes profesionales y anti migratorios sobre la migración de fibroblastos in vitro Esferoides 3D del fibroblasto pueden ser formados vía colgante gota cultura durante un periodo de 72 h (figura 1). Tras el período de la cultura, esferoides se encajan dentro de la matriz del coágulo y reflejada mediante microscopía de campo luminoso (fi...

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

El objetivo de este protocolo es evaluar el efecto de los factores pro y anti migratory en migración de la célula dentro de una matriz tridimensional de fibrina.

Caracterización de la migración de la célula en tres dimensiones In Vitro herida ambientes

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 vistas.  North Carolina State University and The University of North Carolina - Chapel Hill. El objetivo de este protocolo es evaluar el efecto de los factores pro y anti migratory en migración de la célula dentro de una matriz tridimensional de fibrina.

Video: 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.

Tridimensional óptico resolución microscopía fotoacústica

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

Investigación

Bioingeniería

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).

Campo eléctrico controlado por migración dirigida de células progenitoras neurales en entornos 2D y 3D

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.

Microinyección de Heridas Ensayo y<em&gt; In vivo</em&gt; Localización de la herida epidérmica Reporteros de respuesta en<em&gt; Drosophila</em&gt; Embriones.

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.

Análisis de la migración de células dentro de una matriz de colágeno tridimensional

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.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

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).

Irritación de los ojos de prueba (IET) para la identificación del peligro de los ojos: Irrita los productos químicos que utilizan Reconstruido humana Córnea-como epitelial (RHCE) Modelo de Tejidos

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.

Preparación y análisis de<em&gt; In Vitro</em&gt; Tres de mama Carcinoma de dimensiones 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.

Ingeniería tridimensionales epiteliales tejidos embebidos dentro de la matriz extracelular

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.

Cultivo de células de mamífero en andamios tridimensionales péptido

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.

Evaluación de la proliferación del Keratinocyte en dos y tres dimensiones substratos de colágeno de tipo I

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. ...