We are grateful to the other members of the lab for their technical assistance in developing the CSE damage assay, and their support and advice during the development of this assay. In addition, we are grateful to the Holy Cross Fenwick Scholar Committee for their support of Nicholas Cormier, and the Holy Cross College Honors program for their support of Alexander Yeo.

Nota: Para este estudio, las células estromales mesenquimales de pulmón (LR-MSC) a partir de tejido de pulmón distal fueron aislados ya sea en base a su expresión de marcadores de superficie celular (neg CD45, CD31 neg, Sca-1 alto, EpCAM neg) 14,15, usando explante fuera el crecimiento 16, o el uso de la digestión enzimática 17. Adherente LR-MSCs se cultivaron en DMEM con alta glucosa y sin glutamina, 15% FBS, glutamina mM de antibiótico / antimicótico, y 2 1x (de ahora en adelante medios de comunicación completa) y se incubó a 37 ° C, 5% de CO 2. LR-MSCs se pasaron 3-4 veces para asegurar una población de células con características de crecimiento relativamente homogéneas para el ensayo de migración. 1. Preparación de una monocapa confluente <ol><li> Para separar las MSC de placas de cultivo estándar 100 mm, lavar las células con 10 ml de 37 ° C PBS precalentado, aspirar el PBS y añadir 4 ml de tripsina (0,25%). Incubar 3-5 min en una incubadora a 37ºC. Aspirar el cells en un tubo cónico, añadir un volumen igual de medio completo y se centrifuga a 300 g durante 5 min para sedimentar las células hacia abajo. Nota: Longer incubación en tripsina puede alterar los receptores de la superficie celular y potencialmente afectar a la migración. </li><li> Aspirar los medios de comunicación y Resuspender las células en medio completo fresco. Recuento de células, con exclusión de células muertas usando la exclusión de azul de tripano y la placa células estromales mesenquimales 500.000 en 4 ml de medio completo en un 60 mm placa de cultivo celular. Nota: Dependiendo de la fuente de las células, las pruebas pueden ser necesarias para determinar el número óptimo de células necesarias. </li><li> Se incuban las células hasta que las placas son 90% de confluencia para la fijación celular y la proliferación óptima, por lo general de 48-72 h para MSCs. Nota: No se recomienda el 100% de confluencia, ya que muchos de inhibición experiencia células contactos dependiente de anclaje, que puede alterar su capacidad migratoria. Placas con menos de 80% de confluencia se traducirá en fotos inutilizables debido a que el software de análisis de imagen interpretará brechas entre las células que forman parte del perímetro de la cero (ver Figura 1D). </li><li> Después de que las células han alcanzado 90% de confluencia, dañar las células según sea apropiado para la aplicación. Para evaluar el daño celular después de la exposición al humo del cigarrillo extracto soluble (CSE), generará 100% CSE dibujando el humo de 1 Universidad de Kentucky cigarrillo investigación (3R4F) a través de 25 ml de medio completo en un Kitasato más de 2 min 18. </li><li> Se incuban las células durante 24 horas en 4 ml 1.4% CSE diluido en medio completo. Después de la incubación, se lavan las células dos veces con 4 ml de PBS estéril caliente, antes de sustituir con 4 ml de medio completo fresco. Nota: Las células Dañar con altas concentraciones de extracto de humo de cigarrillos (&gt; 5%) pueden reducir sustancialmente confluencia. </li></ol> 2. Creación de Scratch <ol><li> En un ambiente estéril, retire la tapa de la placa de 60 mm de MSC dañados, y sentar un borde recto (por ejemplo, una regla de plástico esterilizada) across el borde superior de la placa. Sin quitar los medios de comunicación, utilizar una punta de pipeta 200 l estéril guiada por la recta de punta estéril para hacer una a cuatro rayas paralelas a través de la placa de aproximadamente 5 mm de distancia y 50 mm de largo. Nota: Un borde recto minimiza la cantidad de ajustes rotacionales necesarias para la placa cuando está en la platina del microscopio. Es imperativo que los arañazos aparecen completamente vertical (u horizontal) en la pantalla. Generación de múltiples arañazos en una placa de 60 mm no altera el microambiente de una manera que afecta a la migración celular (datos no presentados), pero puede ser una consideración para otros tipos de células. No se recomienda el uso de pozos más pequeños ya que la variabilidad de las células que crecen cerca del borde del pozo se hace más pronunciada y puede afectar a la generación uniforme de arañazos. </li><li> Aspirar los medios y lavar suavemente las placas con 2 ml de medio completo pre-calentado para evitar que las células se adhieran a la centro de la cero, y de quitar celulars que se han aflojado por el rascado. Reemplace con 4 ml de medio completo fresco. </li><li> Utilizando un marcador permanente estéril, etiquetar cada arañazo de 1-4 en la parte inferior de la placa en el lugar que no interfiera con las imágenes de los arañazos. Nota: Es importante que las imágenes de la misma cero se comparan antes y después de la migración, ya que no puede haber variación en el ancho cero. </li></ol> 3. Teniendo iniciales Imágenes del rasguño Nota: Si el ensayo cero se realiza en múltiples placas, se recomienda que se tomen las imágenes iniciales de todos los arañazos en un solo plato antes de arañazos se hacen en la siguiente placa. <ol><li> Utilice un microscopio de contraste de fase invertida con una cámara para generar las imágenes necesarias para el análisis. </li><li> Coloque la placa de cultivo en el microscopio, y utilizar el objetivo de baja potencia (10X) para localizar cero # 1. Si la condensación en la tapa de la placa de cultivo está impidiendo una imag clarae de las celdas, utilice un escenario calienta a 37 ° C. </li><li> Mantener el cero totalmente horizontal y en el centro del campo de visión, obtener tantas imágenes como sea necesario para abarcar el 90% de la longitud del cero. Obtener imágenes de partes distintas del cero sin superposición entre las imágenes. Nota: La refracción de la luz en los bordes de la placa de cultivo sobreexpone las imágenes, haciéndolas imposibles de analizar. Por lo tanto, no tome imágenes de la primera y el último 5% de la longitud del cero. </li><li> Guardar imágenes de cada cero en una carpeta separada. </li><li> Repita los pasos 3,2 a 3,4 para los rasguños 2, 3 y 4. </li><li> Placas a la incubadora volver inmediatamente después de la impresión inicial. Deje que las células migran por 4-7 hr. Nota: 7 horas es óptimo cuando se evalúa MSC dañadas con CSE. La incubación de las células durante más de 7 horas no se recomienda debido a la proliferación de células puede actuar como una variable de confusión para la migración celular (véase la nota opcional 8.2 más adelante). </li></ol><pclass = &quot;jove_title&quot;&gt; 4. Análisis iniciales de Scratch Imágenes <ol><li> Descargar ImageJ e instalar el plugin Herramienta Curación de Heridas (consulte la Tabla de Materiales / Equipo). </li><li> Abra el archivo de imagen en ImageJ, haga clic en &quot;Proceso&quot; y &quot;Find Edges&quot; para destacar las células que rodean el cero para la Herramienta de Curación de Heridas para calcular el área de borrador (Figura 2B). A continuación, haga clic en &quot;Imagen&quot;, &quot;Ajuste&quot;, &quot;Umbral de color&quot; y en el menú desplegable llamado &quot;Umbral de color&quot; cambiar el valor a &quot;B &amp; W&quot;. </li><li> Dentro del menú &quot;Umbral de color&quot;, ajuste ambos controles deslizantes de umbral de luminosidad hasta conseguir un contraste óptimo entre el cero y celular frente. Para permitir la mayor rango cuando se trata de crear un contraste en la imagen, mueva el control deslizante inferior completamente a la derecha (imagen completamente negro), y luego ajuste el control deslizante superior para generar el máximo contraste. </li><li> Lentamente ajustar el bri superiordeslizador llo de izquierda a derecha hasta que la imagen muestra limpiamente el contorno del cero (Figura 2C). Una vez se han identificado los bordes de la cero, haga clic en &quot;Más Herramientas&quot; en la barra de herramientas ImageJ, seleccione &quot;MRI_Wound_Healing_Tool&quot; en el menú desplegable y pulse el botón &quot;m&quot; (que aparecerá en la barra de herramientas ImageJ) para resaltar la zona cero y la salida de la zona calculada en los resultados de una ventana pop-out (Figura 2D). </li><li> Copie y pegue todos los números de la ventana de resultados (Figura 2E) en una hoja de cálculo Excel. Asegúrese de separar los datos de arañazos individuales. </li></ol> 5. Teniendo Finales Imágenes del rasguño <ol><li> Después de que las células han migrado (07.04 h), repita los pasos 3,2-3,4. Imagen de las placas en el mismo orden en que fueron rayados por lo que las células en cada placa han tenido el mismo tiempo a migrar. Nota: Las células se pueden obtener imágenes en múltiples puntos de tiempo, dependiendo de su sensitividad tanto a los cambios de temperatura y pH. Alternativamente, imágenes de células vivas se puede utilizar para seguir las células en tiempo real a medida que migran. </li></ol> 6. Analizar finales de Scratch Imágenes <ol><li> Repita los pasos 4,2-4,5 para imágenes Final Scratch. </li></ol> 7. Cálculo del área de borrador para cuantificar las Migraciones <ol><li> Calcule el área media previa a la migración promediando los valores de área iniciales de los respectivos arañazos. </li><li> Calcular la cantidad total de la migración restando el área de la migración final para cada imagen, calculado en el paso 6, de la zona de pre-migración promedio (paso 7.1). Generar una lista de valores para cada cero que representa el cambio en la migración. </li><li> Agrupar los datos de varios rasguños dentro del mismo grupo de prueba (es decir, todas las placas y todos los rasguños en las células expuestas a 0% CSE). Utilice un análisis de varianza de prueba para analizar las diferencias entre los grupos experimentales. </li><li> Si las células son lentos para migrary requieren un tiempo de incubación más largo después del rascado (mayor a 10-12 hr), realizan un ensayo de incorporación de BrDU o EdU 19 para determinar si las células están experimentando cualquier proliferación durante el periodo en el que se está produciendo el ensayo de migración. Elija un momento ensayo de migración para evitar la proliferación celular significativa. </li></ol> 8. Opcional <ol><li> Para evaluar si la senescencia celular significativa se produce como resultado del procedimiento de rascado, lleve a cabo un ensayo de beta-galactosidasa asociada a la senescencia en una placa de ensayo en momentos predeterminados después de rascarse 19. </li></ol>

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The authors declare that they have no competing financial interests.

El protocolo descrito aquí proporciona un método cuantitativo robusto, estandarizado para realizar y analizar los ensayos de cero. Los ensayos de cero simples se utilizan rutinariamente en muchas áreas diferentes de estudio para examinar la migración celular. Sin embargo, tradicionalmente el ensayo cero ha carecido de una puesta a punto y cuantificación protocolo estandarizado, lo que ha dado lugar a problemas con la reproducibilidad 7,10. Muchas de las modificaciones y optimizaciones para mejorar el ens...

La migración celular es altamente coordinada y vital para muchos procesos fisiológicos tales como el desarrollo de tejidos, la reparación y la regeneración, así como procesos patológicos tales como la metástasis del cáncer y la arteriosclerosis 1. Una comprensión de la migración celular es particularmente relevante para las nuevas terapias utilizadas para reparar los tejidos dañados y tratar condiciones patológicas, incluidas las tecnologías de trasplante de células y tejido artificial injerto 2. Dado el interés actual en el papel de las células estromales mesenquimales (MSC) en la mediación de la reparación de tejidos 3, la cuantificación de la capacidad migratoria de estas células utilizando un ensayo que es rigurosamente cuantificables, adaptable y rentable es de interés. Es importante destacar que dicho ensayo deberá ser lo suficientemente sensible para detectar cambios relativamente sutiles en la capacidad migratoria celular después del daño. Los métodos actuales para cuantificar la migración de células, incluyendo los ensayos de cero, los ensayos de migración trans-pocillos (Boyden chámbar), matrices microcolumnas, y ensayos de la zona de exclusión de células poseen una serie de limitaciones en la reproducibilidad, personalización, cuantificación, y la rentabilidad 1,4,5. El ensayo cero optimizado descrito aquí demuestra resultados robustos, capacidades de análisis cuantificables y basados ​​en imágenes, la rentabilidad y la capacidad de adaptación a otras aplicaciones. Los ensayos de cero se han utilizado en múltiples capacidades para evaluar la migración y la proliferación celular en diferentes condiciones experimentales 5. El ensayo implica la siembra de las células designadas, el crecimiento de ellos para completar o cerca de confluencia, y arañar la monocapa resultante con una aguja estéril o punta de la pipeta 6. Análisis es más comúnmente lleva a cabo comparando el ancho de la cero a través de múltiples puntos de tiempo en lugares seleccionados al azar 7-9. A pesar de su uso prominente, el ensayo cero es confundida por problemas con la reproducibilidad y la cuantificación. La variabilidad engeneración del cero no sólo altera el microambiente de las células, pero también puede impedir la migración de células al dañar la superficie de la placa y la matriz extracelular subyacente 5. Los ensayos se llevan a cabo con frecuencia más de 7 a 12 horas, sin embargo, para las líneas celulares que muestran la migración más lenta y tiempos de ensayo más largos, la proliferación se convierte en una variable de confusión 7,10. Por último, las células senescentes generados por el proceso de rascado pueden liberar factores que interfieren con la señalización extracelular requerida para cerrar la brecha en la monocapa 1. Optimización del ensayo de cero requiere la creación de un vacío constante que no interfiera con las propiedades de superficie, minimizando ensayo de duración de tiempo, y la prevención de la muerte celular no deseada durante la manipulación. El ensayo basado tapón es una optimización de un ensayo de zona de exclusión celular. Este ensayo utiliza un tapón colocado en el centro del pozo que excluye el crecimiento celular, pero permite que las células se sembraron alrededor de la zona de exclusión central.Para evaluar la migración, se quita el tapón, y la zona de exclusión resultante proporciona una superficie para que se produzca la migración. Sin embargo, este ensayo es difícil personalizar o adaptar 10 y para algunas aplicaciones, esta técnica también puede tener un costo prohibitivo. En contraste con los ensayos de cero y sus derivados, los ensayos de migración trans-bien (o ensayos de cámara de Boyden) evaluar la migración mediante la cuantificación del número de células que se mueven de una cámara, a través de una membrana de filtro microporoso, en una cámara que contiene agentes quimiotácticos 8,11, 12. Esta técnica tiene una utilidad limitada para las células adherentes como MSCs porque después de la migración a través de la membrana porosa, las células se adhieren a un lado membrana expuesta a los agentes quimiotácticos, y pueden ser difíciles de cuantificar con precisión. Mientras que el ensayo es capaz de examinar algunos patrones de migración en tres dimensiones, los tipos de células restringidas de las que es capaz de cuantificar con precisión el límite de migración celular su utilidad 10. Otra alternativa a los ensayos de cero utiliza una matriz de micro-pilar, que mide la motilidad celular a través de un espacio tridimensional utilizando la capacidad de las células para deformar y migrar en la matriz como un sustituto. Polydimethlysiloxane (PDMS) elastómeros curados sobre un molde preciso y tratados con ozono y fibronectina produce un microambiente homogénea y no degradable. Espaciamiento Micro-pilar también se puede variar como sea necesario para medir la capacidad de las células para entrar en la matriz 4. El molde se crea a través de grabado iónico reactivo profundo de obleas de silicio para crear una versión negativa de la matriz de alta relación de aspecto 13. Mientras que el ensayo se refuerza por su personalización, capacidad de modelar la migración tridimensional, y el análisis a través de la visualización directa de células que migran, dificultad en la creación de matrices de micro-pilar económicamente impide su uso generalizado. El ensayo cero optimizado descrito en este protocolo proporciona un eficiente, cosmétodo t-efectiva para producir rasguños consistentes que pueden ser analizados utilizando el software de libre acceso. En lugar de medidas de ancho simples hechas a través del cero antes y después de la migración celular, el software permite al usuario determinar las áreas totales de arañazos antes y después de la migración. Este avance limita el problema de tratar de determinar dónde está el cero las medidas de ancho se deben tomar, y si el ancho de la cero es uniforme a lo largo de su longitud. Además, la optimización cuidadosa del número de células, la confluencia celular y el tipo y grado de daño causado a las células se discute con el fin de optimizar aún más el ensayo.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">DMEM (high glucose, no added glutamine)</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">31053-036</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fetal Bovine Serum, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3091002</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Antibiotic/antimycotic, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SV3007901</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:527px;">Glutamine, 200mM, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3003401</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">TypeLE cell dissociation reagent</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">12563-01</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Dulbecco&#39;s Phosphate Buffered Saline, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3002802</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Falcon standard 60mm tissue culture dishes</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">08-772B</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fisherbrand Sterile 200ul pipet tips</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td>02-707-502</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Inverted light microscope with camera attachment</td>
			<td style="width:221px;">Variable</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:527px;">ImageJ software&#160;</td>
			<td style="width:221px;">http://imagej.nih.gov/ij/</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:527px;">MRI_Wound_Healting plugin</td>
			<td style="width:221px;"><a href="http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool">http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool</a></td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Statistical analysis software</td>
			<td style="width:221px;">Microsoft Excel</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
	</tbody>
</table>

optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

Los ensayos de cero aquí presentados se realizaron con residentes de pulmón de células estromales mesenquimales murinos (LR-MSC) y se aislaron como se indica en las notas de protocolo. La LR-MSC se sembraron a una densidad de 500.000 células en un 60 mm placa de cultivo tisular, y se cultivaron hasta 90% de confluencia en 48 hr. Para generar daños, 4% CSE (descrito anteriormente) se incubó con las células durante 24 hr después del periodo de siembra inicial pero antes de que el ensayo cero. Para cada ensayo, el ...

Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Optimización del ensayo arañazos herida para detectar cambios en Murino mesenquimales estromales migración de células después de daño por Soluble Extracto humo del cigarrillo

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

optimization-wound-scratch-assay-to-detect-changes-murine-mesenchymal

Research

JoVE Journal

Developmental Biology

16.8K vistas.  College of the Holy Cross. The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Video: Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Pez cebra queratocitos explantes para estudiar la migración celular colectiva y reepitelización en Curación de Heridas cutáneas

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Investigación

Biología del Desarrollo

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Evaluación de la migración de células endoteliales después de la exposición a sustancias químicas tóxicas

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Desarrollo de una<em&gt; In Vitro</em&gt; Ensayo para evaluar la función contráctil de las células mesenquimales que la transición epitelio-mesenquimal Sufrió

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Aislamiento y expansión de células madre mesenquimales / estromales derivadas de tejido de placenta humana

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Producción y Administración de la terapéutica madre mesenquimales / célula del estroma (MSC) esferoides imprimado en 3-D Los cultivos bajo condiciones Xeno-libres

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Aislamiento y caracterización de las células mesenquimales del estroma de cordón umbilical humano y fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

Un dispositivo para realizar la migración de células / Curación de Heridas en una placa de 96 pocillos

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

Un ensayo de transferencia de puntos de 5 mC cuantificando el nivel de metilación de ADN de la distredenciación de condrocitos<em&gt; In Vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

El ensayo de co-cultivo del anillo aórtico: Una herramienta conveniente para evaluar el potencial angiogénico de las células mesenquimales estromales In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Visualización de la migración celular tangencial en el Tectum óptico de Chick en desarrollo

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...