Los autores no tienen ninguna agradecimientos.

1. preparación de las células
<ol><li>Placa de 20.000 células en 2 mL de medio de cultivo en cada pocillo de una placa de 6 pozos utilizando una técnica estéril en un gabinete de bioseguridad.</li>
	<li>Colocar placas de 6 pocillos en una incubadora humidificada (37 ° C, 5% CO2) durante 24 h permitir que las células se adhieren a las placas.</li>
</ol>2. preparación del fotosensibilizador para el tratamiento de las células
<ol><li>Preparar soluciones de 0.5, 1 y 2 mM 5-ALA en medio de cultivo. Si el suero bovino fetal (FBS) es un ingrediente del medio de cultivo, preparar y tratar las células con 5-ALA en la solución de medio de cultivo con 0,1% FBS para incubaciones. 11 , 12 en este caso, las células SCC-13 no requieren FBS, así 5-ALA fue añadido directamente a queratinocitos medio libre de suero suplementado con extracto de pituitaria bovina y factor de crecimiento epidérmico.</li>
	<li>Aspirar el medio de cultivo y lavar las células con al menos 2 mL de tampón fosfato salino (PBS).</li>
	<li>Aspire PBS y añadir 2 mL de las soluciones de 5-ALA 0, 0,5, 1 o 2 mM en placas separadas o pozos.</li>
	<li>Incubar las células con 0, 0.5, 1 o 2 mM 5-ALA en una 36 a 37 ° C calefacción bloque 30 min proteger las células de la exposición a la luz durante la incubación con papel de aluminio.</li>
	<li>Las soluciones de 5-ALA 0, 0.5, 1 y 2 mM de aspirar y lavar las células con 2 mL de PBS.</li>
	<li>Aspire PBS y añadir 2 mL de medio de cultivo para la irradiación de la luz azul.</li>
</ol>3. azul fototerapia luz
<ol><li>Antes de irradiar las células, activar el aparato de luz azul (p. ej., diodo emisor de luz, fluorescente o halógena) y duración de un ciclo (1.000 s) para calentar el dispositivo. El dispositivo de luz azul debería tener una longitud de onda de salida de 417 +/-5 nm. Permitiendo que el dispositivo funcione durante un ciclo antes de tratamientos se asegura que la longitud de onda de luz azul y la irradiación son constantes a lo largo de la fase de fotoactivación.</li>
	<li>Utilice un fotómetro para medir la irradiancia en la superficie celular. La irradiación de luz azul en la superficie de la célula debe ser 10 mW/cm2. La distancia entre la luz y las células deba ajustarse para lograr una radiación de 10 mW/cm2 según la intensidad de la fuente de luz.</li>
	<li>Coloque grupos de tratamiento ALA 0, 0.5, 1 y 2 mM sobre una superficie negra debajo de la fuente de luz azul. Irradian las células con luz azul para 1.000 s (16 min y 40 s) para un fluence total de 10 J/cm2. Después de la fotoactivación luz azul, las células están listas para análisis.</li>
</ol>4. colección y tinción
<ol><li>Preparar el búfer de flujo según las instrucciones del fabricante (véase Tabla de materiales).</li>
	<li>Aspirar el medio de cultivo y lavado las células con 2 mL de PBS.</li>
	<li>Añadir 1 mL de 0.25% tripsina-EDTA y permiten separar por aproximadamente 3 a 5 min de las células. Las células pueden ser examinadas bajo el microscopio para confirmar la separación de la célula.</li>
	<li>Añadir 1 mL de medio de cultivo (o PBS) con 10% suero fetal bovino para desactivar la tripsina.</li>
	<li>Recoja la suspensión celular en tubos de flujo etiquetado 5 mL.</li>
	<li>Girar el tubo de flujo de 5 mL en una centrifugadora a 201 x g por 5 min aspirar solución sin remover el sedimento celulares.</li>
	<li>Agregar 200 μL de tampón de flujo con anticuerpo conjugado de annexin-V (1 μl de annexin-v por 39 μl de tampón de flujo) para cada muestra.</li>
	<li>Resuspender las células y en la incubadora (37 ° C, 5% CO2) por 20 min permitir la Unión de la anexina V.</li>
	<li>Agregar 3 μl de 7-AAD a cada muestra. Incubar por 5 min a temperatura ambiente.</li>
</ol>5. el análisis cytometric del flujo de
<ol><li>Siga instrucciones de fabricante de citómetro de flujo para la disposición (véase Tabla de materiales).</li>
	<li>Recoger y analizar muestras con citometría de flujo. 13
</li>
	<li>Realizar una comparación estadística de los grupos de tratamiento y control mediante análisis de varianza (ANOVA)14. Comparar las medias de los grupos de tratamiento a la media del control con la prueba de Dunnett para análisis post hoc .</li>
</ol>

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C., Lou, X., Zhang, Z., Ingram, P., Yoon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cancer+cell+sphere+formation+for+characterizing+the+efficacy+of+photo+dynamic+therapy+in+3D+cell+cultures.">High-throughput cancer cell sphere formation for characterizing the efficacy of photo dynamic therapy in 3D cell cultures.</a> Scientific Reports. 5, 12175 (2015).</li><li>Campos, C., Inada, N., Kurachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+PDT+on+breast+cancer+spheroids.">Low-dose PDT on breast cancer spheroids.</a> Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXVII. , (2018).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+temperature+on+5-aminolevulinic+acid-based+photodynamic+reaction+in+keratinocytes+in+vitro.">The influence of temperature on 5-aminolevulinic acid-based photodynamic reaction in keratinocytes in vitro.</a> Photodermatology, Photoimmunology &amp; Photomedicine. 26 (2), 83-88 (2010).</li><li>Juzeniene, A., Juzenas, P., Kaalhus, O., Iani, V., Moan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+Accumulation+of+protoporphyrin+IX+after+topical+application+of+5-aminolevulinic+acid+and+its+methylester+and+hexylester+derivatives+in+normal+mouse+skin.">Temperature effect on Accumulation of protoporphyrin IX after topical application of 5-aminolevulinic acid and its methylester and hexylester derivatives in normal mouse skin.</a> Photochemistry and Photobiology. 76 (4), 452-456 (2002).</li><li>Novak, B., Heesen, L., Schary, N., Lubbert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+different+illumination+parameters+on+protoporphyrin+IX+induced+cell+death+in+squamous+cell+carcinoma+cells.">The influence of different illumination parameters on protoporphyrin IX induced cell death in squamous cell carcinoma cells.</a> Photodiagnosis and Photodynamic Therapy. 21, 385-392 (2018).</li></ol>

DUSA/Sun farmacéutica proporciona 5-ALA y el dispositivo de luz BLU-U. Este manuscrito fue apoyado por residual de Dr. Jagdeo financiación. Los contenidos no representan las opiniones del Departamento de Asuntos Veteranos de Estados Unidos o el gobierno de Estados Unidos.

Hubo un aumento significativo en la apoptosis de SCC-13 tras incubaciones de 30 min 0.5, 1 y 2 mM 5-ALA siguieron por 1.000 s de luz azul. Estos resultados son consistentes con nuestra investigación previamente publicado demostrando que incubaciones de 5-ALA de 30 min, seguida de activación luz azul conduce a un aumento dosis dependiente en el porcentaje de fibroblastos células que experimentan apoptosis12, 14.
Este enfoque experimental para estudiar PDT tiene ventajas y limitaciones. Los métodos descritos se ajustan a la práctica clínica como un PS comercialmente disponible y se utilizaron fuente de luz. Los investigadores pueden personalizar los métodos con diferentes tipos de células, PSs, fuentes de luz y parámetros de la irradiación. Sin embargo, existen limitaciones a este enfoque como este protocolo está diseñado para células adherentes en una monocapa. En la práctica clínica, las diferencias en arquitectura o enfermedad patología del tejido (es decir, hiperqueratosis o fibrosis) pueden disminuir la absorción de PS o penetración15. Como resultado, las dosis de tratamiento y los resultados experimentales no directamente correspondan a la práctica clínica. Modelos de tumores esferoide y chip de microfluidos se han estudiado métodos para replicar más de cerca tumor microambientes16,17,18. Esferoides tienen nichos celulares internas y externas que pueden incorporar el photosensitizers diferencialmente y representan arquitectura tumor heterogéneo. Otros investigadores han utilizado chips microfluídicos para condiciones de tratamiento de la pantalla y suministro vascular de photosensitizers. Sin embargo, chips microfluídicos pueden ser costosos de desarrollar o difícil de aplicar para los investigadores que no estén familiarizados con la técnica. Además, esferoides y chips microfluídicos pueden no reflejar el tratamiento clínico de los cánceres de piel en la cual photosensitizers se aplican directamente a la superficie del tumor sin diseminación vascular. Los investigadores deben evaluar los pros y contras de la cultura de monocapa, modelos de tumores esferoide y chips microfluídicos para estudiar PDT en sistemas diferentes de enfermedades. Como no existen células inmunes o matriz extracelular, no es posible determinar cómo afecta a la PDT las interacciones célula-célula compleja. Los investigadores deba confirmar en vitro resultados experimentales utilizando modelos animales y ensayos clínicos. Para lograr alta validez de la prueba, es importante mantener la incubación constante y luz de parámetros al realizar experimentos en vitro PDT.
Temperatura se sabe que alteran la eficacia de la PDT19. Un estudio demostró que la muerte celular, absorción del 5-ALA, PP IX formación y liberación de citoquinas pueden mejorar incubando células con 5-ALA en temperaturas hasta 44 ° C19. Temperatura de incubación superior a 44 ° C puede llevar a la muerte celular inducida por termales, y temperaturas de incubación por debajo de 20 ° C no pueden llevar a la absorción significativa de 5-ALA y acumulación19. Se recomienda que los investigadores realizan incubaciones de PS en un bloque de calefacción regulable en temperatura en una temperatura constante con control de confusión potencialmente efectos térmicos. Además, es importante incubar el fotosensibilizador para una cantidad suficiente de tiempo para permitir la conversión de 5-ALA en PP-IX. En este protocolo, se incubaron las células SCC-13 con 5-ALA durante 30 minutos, que con éxito indujo la apoptosis de las células. Previamente hemos demostrado que una incubación de 10 min de 5-ALA no aumentó significativamente la apoptosis en el fibroblasto en comparación con el control sin tratar fibroblastos11,14. PP-IX empieza a acumular en la piel del ratón después de 5-ALA es incubar durante 6 minutos a 37 ° C. Por lo tanto, después de diez minutos de incubación de 5-ALA, puede no haber suficiente acumulación de PP-IX para inducir la apoptosis de la célula20. Se recomienda un período de incubación mínimo de 20 a 30 min de 5-ALA basado en nuestros anteriores estudios11,14. Los investigadores deben optimizar el período de incubación basado en el laboratorio de ajuste, fotosensibilizador de interés, tipo de la célula y la indicación clínica. Experimentos de optimización y valoración de anticuerpos pueden realizarse para obtener mejores resultados utilizando las directrices de los fabricantes. Otros ensayos de interés pueden realizarse después de la fotoactivación luz azul incluyendo cuantificación de citometría de flujo dihydroethidium de ROS. 14
Variación en la cantidad de luz entregada por unidad de superficie (es decir, radiación) y dosis total de irradiación (es decir, fluence) durante la fase de fotoactivación pueden afectar la formación de ROS y de la célula apoptosis21. La relación entre la fluencia, irradiancia y tiempo puede ser descrita por la siguiente ecuación:
Fluencia (J/cm2) = irradiancia (W/cm2) x tiempo (s)
Como fluence es dependiente en el tiempo, alargando o acortando las fases de fotoactivación puede cambiar la eficacia del tratamiento. La radiación es proporcional a la distancia al cuadrado entre la fuente de luz y el tejido diana. Como resultado, si la luz está demasiado lejos, puede no haber suficiente energía de la luz entregado a los tejidos específicos para excitar el PS. alternativamente, se puede aumentar la irradiación por la luz que refleja de las superficies que rodean. Por lo tanto, se recomienda realizar irradiaciones de luz con las placas de cultivo celular colocadas sobre una superficie negra para evitar la reflexión de la luz. Los investigadores pueden adquirir comercialmente disponibles o aprobados por la FDA azul diodos emisores de luz y dispositivos fluorescentes para utilizar en experimentos de PDT, pero estos dispositivos tienen diferentes potencias y uniformidad de campo de la luz. Un fotómetro de longitud de onda específica debe utilizarse para medir la irradiancia en la superficie de la célula y la uniformidad del campo ligero antes de cada experimento para obtener resultados consistentes. Un difusor disponible comercialmente puede utilizarse para mejorar la uniformidad del campo, si es necesario.
En Resumen, hemos descrito un enfoque en vitro a investigar PDT por detalle teoría, métodos experimentales, limitaciones, peligros potenciales y las recomendaciones para la optimización. PDT es que una investigación clínica útil del procedimiento y en vitro puede permitir el desarrollo de la novela PSs, optimización de protocolos y nuevas indicaciones de PDT.

Terapia fotodinámica (TFD) es un procedimiento médico que involucra la incubación de un fotosensibilizador exógeno aplicado (PS) seguido por fotoactivación luz visible para inducir la apoptosis de las células. La Federal Drug Administration (FDA) ha aprobado el PDT para el tratamiento de queratosis actínica (AK), y las guías clínicas recomiendan PDT como tratamiento para algunos cánceres de piel no melanoma y el acné vulgaris1,2. Usos emergentes no dermatológicas para PDT incluyen el tratamiento de cánceres ginecológicos, próstata y gastrointestinal3. PDT es una modalidad terapéutica ventajosa ya que es bajo costo, no invasiva y asociada con mínimos efectos adversos y cicatrices2.
En el primer paso de la TFD, PS se aplica y permite la acumulación intracelular2. En los Estados Unidos, el ácido 5-aminolevulínico tópico (5-ALA) se utiliza comúnmente para tratar las AKs. Durante la incubación fase, 5-ALA se convierte en protoporfirina IX (PP-IX) a través de la ruta de biosíntesis de hemo en células incorporado3. Las células de cáncer y pre-han disminuido la actividad del ferrochelatase, que convierte la protoporfirina IX (PP-IX), en el producto final, heme. Como resultado, las células de cáncer acumulan PP-IX en comparación con las células normales4,5. Esta acumulación de PP-IX en el cáncer y las células precancerosas en comparación con el tejido circundante permite PDT a ser un enfoque orientado con mínimos efectos adversos. Irradiación de la luz conduce a oxígeno reactivo (ROS) las especies generación cuando el oxígeno acepta electrones excitados del PP-IX. PP-IX tiene un máximo de absorción en el espectro ultravioleta con pequeños picos en el espectro de luz visible, incluyendo azul y rojo luz2. Formación de ROS en última instancia puede conducir a la apoptosis celular, disrupción de la membrana, daño mitocondrial, modulación inmune, proliferación del keratinocyte y colágeno volumen6,7,8,9 , 10.
PDT se desarrolló en la década de 1970 y es una evolución de la modalidad terapéutica3. El protocolo aprobado por la FDA para el tratamiento de AKs recomienda incubación 5-ALA piel de 14 – 18 h, pero incubaciones de 1 a 2 h se utilizan en la práctica clínica2. In vitro, hemos demostrado que incubaciones de 5-ALA de 15 minutos más cortas pueden incrementar la apoptosis de las células del fibroblasto en comparación con los fibroblastos11,12. Además, se están estudiando chlorin novela, ftalocianina y PSs basados en nanopartículas para mejorar la eficacia de PDT3. Adjunto, presentamos un método en vitro para estudio PDT en un carcinoma de célula squamous adherente de las células. En este protocolo, se incuban las células SCC-13 con 0, 0.5, 1.0 y 2 mM 5-ALA de 30 min y fotoactivado con luz 417 nm azul 1.000 s. Medida de resultado primaria es apoptosis y necrosis, medido por citometría de flujo de annexin-V y aminoactinomycin 7 D (7-AAD). Este protocolo de tratamiento está diseñado para simular el PDT y puede ajustarse a estudiar el uso de la TFD con otras líneas celulares, photosensitizers, temperaturas de incubación o longitudes de onda de fotoactivación. Preparación de grupos de control adicional puede ser necesario, incluyendo el fluoróforo sin mancha, solo manchado, controles positivos y negativos para citometría de flujo. Una revisión exhaustiva de la teoría, protocolos, diseño experimental y compuerta para citometría de flujo necrosis apoptosis se puede encontrar en otra parte13.

<table border="0" cellpadding="0" cellspacing="0" width="1259">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Keratinocyte serum free medium&#160;</td>
			<td style="width:211px;">Thermofisher</td>
			<td style="width:204px;">17005042</td>
			<td style="width:623px;">Culture medium for SSC-13 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM low glucose glutamax</td>
			<td>Thermofisher</td>
			<td>10567022</td>
			<td>Possible culture medium for other cell types</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">6-well culture plates</td>
			<td>Thermofisher</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Thermofisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Thermofisher</td>
			<td>15250061</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLU-U light&#160;</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td>Other blue LED, flourescent, or halogen lights may used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ALA</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta Biologicals</td>
			<td>C17032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowCellect Annexin Red Kit</td>
			<td>Millipore Sigma&#160;</td>
			<td>FCCH100108&#160;</td>
			<td>Propidium iodide may replace 7-AAD. Comes with annexin-V, 7-AAD, and flow buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCanto II</td>
			<td>BD</td>
			<td>Request from manufacturer</td>
			<td>Any flow cytometer may be used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counting Chamber</td>
			<td>Hausser</td>
			<td>3120</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo</td>
			<td>FlowJo</td>
			<td>Request from manufacturer</td>
			<td>Flow cytometry analysis software</td>
		</tr>
	</tbody>
</table>

an in vitro approach to photodynamic therapy

Terapia fotodinámica (TFD) es un procedimiento médico que involucra la incubación de un fotosensibilizador exógeno aplicado (PS) seguido por fotoactivación luz visible para inducir la apoptosis de las células. La Federal Drug Administration ha aprobado el PDT para el tratamiento de la queratosis actínica, y guías de práctica clínicas recomiendan PDT como tratamiento para algunos cánceres de piel no melanoma y Acné vulgaris. PDT es una modalidad terapéutica ventajosa ya que es bajo costo, no invasiva y asociado con efectos adversos mínimos y asustando. En el primer paso de la TFD, PS se aplica y permite la acumulación intracelular. Posterior irradiación de la luz induce la formación de especies reactivas del oxígeno, que en última instancia puede conducir a la apoptosis celular, disrupción de la membrana, daño mitocondrial, modulación inmune, la proliferación del keratinocyte y facturación de colágeno. Adjunto, presentamos un método en vitro para estudio PDT en una línea de células adherentes. Este protocolo de tratamiento está diseñado para simular el PDT y puede ajustarse a estudiar el uso de la TFD con distintas líneas celulares, photosensitizers, temperaturas de incubación o longitudes de onda de fotoactivación. Carcinoma de células escamosas las células se incubaron con 0, 0.5, 1.0 y 2 mM de ácido 5-aminolevulínico (5-ALA) durante 30 min y fotoactivado con luz 417 nm azul 1.000 s. Medida de resultado primaria fue apoptosis y necrosis, medido por citometría de flujo D anexina V y 7 aminoactinomycin. Hubo un aumento dependiente de la dosis en la apoptosis de la célula después de 30 minutos incubación de 5-ALA. Para lograr alta validez de la prueba, es importante mantener la incubación constante y luz de parámetros al realizar experimentos en vitro PDT. PDT es que una investigación clínica útil del procedimiento y en vitro puede permitir el desarrollo de la novela PSs, optimización de protocolos y nuevas indicaciones de PDT.

Después de SCC-13 las células fueron incubadas durante 30 minutos con 0, 0.5, 1 y 2 mM 5-ALA e irradiadas con 1.000 s de luz, hubo un aumento dependiente de la dosis en la apoptosis celular azul. Total apoptosis (media ± error de estándar de la media) era de 3.94 ± 0,34, 7.90 ± 0,52, 23.86 ± 0,52 y 38.33 ± 1.81 después de 30 min de incubación con 0, 0.5, 1 y 2 mM 5-ALA, respectivamente y 1000 segundos de azul la luz (figura 1). Comparamos el porcentaje medio de células apoptóticas entre 0, 0.5, 1 y 2 mM 5-ALA incubado grupos mediante ANOVA. Las células tratadas con 1 y 2 mM 5-ALA tenían un aumento significativo en la apoptosis de la célula en comparación con las células tratadas de 0 mM 5-ALA. Figura 2A muestra representativa flujo cytometric gating de forward scatter (FSC) versus dispersión lateral (SSC) SCC-13 células incubadas con 0, 0.5, 1 y 2 mM 5-ALA. La compuerta del círculo encierra la población de células SCC-13. Para este experimento, se recogieron 7500 eventos y la puerta de población de células SCC-13 capturados por lo menos el 90% de los eventos. Figura 2B muestra un diagrama representativo de la 7-AAD en el eje x y la anexina V en el eje y. Las poblaciones celulares son bloqueadas en cuatro cuadrantes (Q1 a Q4) discriminar células apoptóticas. Células de Q1 (alta anexina v, bajo 7-AAD) representa que experimenta apoptosis temprana. Representan células de Q2 (alta anexina v, alta 7-AAD) sometidos a finales apoptosis o necrosis. Representa células de Q3 (baja anexina v, alta 7-AAD) que experimentan necrosis. Representa de Q4 (baja anexina v, bajo 7-AAD) las células que no experimenta apoptosis o necrosis. Para calcular el porcentaje de células que experimentan apoptosis, hemos añadido el porcentaje de células en Q1 y Q2. Discrepancia en longitud de período de incubación de 5-ALA, temperatura de incubación o fotoactivación dosis puede alterar la eficacia del PDT. Figura 3 muestra una representativa parcela citometría flujo anexina V y 7-AAD cuando se varía la temperatura de incubación (es decir, 27, 36 y 42 ° C). Hubo un aumento dependiente de la temperatura en la apoptosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58190/58190fig1.jpg"> Figura 1: incremento dosis dependiente en la apoptosis después de 30 minutos de incubación de 5-ALA. 5-ALA se incubaron a 36 ° C durante 30 minutos seguidos de 1.000 s de luz azul en las células SCC-13. Las barras representan el promedio porcentuales annexin-V las células positivas en cada tratamiento y grupo control. Experimentos se realizaron por triplicado técnica. Significación estadística se determinó mediante ANOVA, p < 0.05 indicada con un asterisco (*). Barras de error representan el error estándar de media ± de la media. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58190/58190fig2.jpg"> Figura 2: Representante flujo Cytometry parcelas. (A) representante de dispersión hacia adelante vs lado dispersión citometría parcelas en unidades arbitrarias (UA) para ejes x e y - 0, 0.5, 1 y 2 mM 5-ALA, las células incubadas. Para este experimento, se recogieron 7500 eventos y la puerta de población de células SCC-13 había capturado el 90% de los eventos. (B) representante de annexin-V vs 7-AAD flujo cytometry parcelas en AU para ejes x e y - 0, 0.5, 1 y 2 mM 5-ALA, las células incubadas. Q1: temprana las células apoptotic, Q2: finales células apoptóticas/necrótico, Q3: células necróticas y Q4: células viables. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58190/58190fig3.jpg"> Figura 3: 5-ALA en diferentes temperaturas de incubación puede alterar la eficacia de la PDT. Representante de annexin-V vs citometría de flujo 7-AAD parcelas en AU para ejes x e y - SCC-13 células incubadas con 0,5 mM 5-ALA en 27, 36 y 42 ° C. Q1: temprana las células apoptotic, Q2: finales células apoptóticas/necrótico, Q3: células necróticas y Q4: células viables. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about An In Vitro Approach to Photodynamic Therapy at JoVE.com

An In Vitro Approach to Photodynamic Therapy

Terapia fotodinámica (TFD) es un procedimiento médico que involucra la incubación de un fotosensibilizador exógeno aplicado (PS) seguido por fotoactivación luz visible para inducir apoptosis. Presentamos un protocolo en vitro PDT diseñado para simular el PDT que puede utilizarse para estudiar las diferencias en incubación de PS y parámetros de tratamiento de la luz.

Un enfoque In Vitro de la terapia fotodinámica

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

an-in-vitro-approach-to-photodynamic-therapy

Research

JoVE Journal

Medicine

An In Vitro Approach to Photodynamic Therapy

8.6K vistas.  University of California, Davis. Terapia fotodinámica (TFD) es un procedimiento médico que involucra la incubación de un fotosensibilizador exógeno aplicado (PS) seguido por fotoactivación luz visible para inducir apoptosis. Presentamos un protocolo en vitro PDT diseñado para simular el PDT que puede utilizarse para estudiar las diferencias en incubación de PS y parámetros de tratamiento de la luz.

Video: An In Vitro Approach to Photodynamic Therapy

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Un In Vitro Sistema para el Estudio de latencia del tumor y el salto hacia el crecimiento metastásico

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Investigación

Medicina

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuevo enfoque quirúrgico para la administración intratraqueal de agentes bioactivos en un modelo de ratón fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando cuantitativa en tiempo real PCR para determinar quimerismo de células donantes en un modelo competitivo Trasplante de médula ósea murina

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

Citotóxica eficacia de la terapia fotodinámica en células de osteosarcoma<em&gt; In Vitro</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can experience a range of short and long term neurobehavioral symptoms (somatic, cognitive and emotional/behavioral) that can have a significant impact on one&#8217;s participation in daily activities and pursuits of interest (e.g., school, sports, work, family/social life, etc.). Despite this, there remains a paucity in clinically driven research aimed specifically at exploring concussion within the youth sport population, and more specifically, multi-modal approaches to measuring recovery. This article provides an overview of a novel and multi-modal approach to measuring recovery amongst youth athletes following concussion. The presented approach involves the use of both pre-injury/baseline testing and post-injury/follow-up testing to assess performance across a wide variety of domains (post-concussion symptoms, cognition, balance, strength, agility/motor skills and resting state heart rate variability). The goal of this research is to gain a more objective and accurate understanding of recovery following concussion in youth athletes (ages 10-18 years). Findings from this research can help to inform the development and use of improved approaches to concussion management and rehabilitation specific to the youth sport community.

This article provides an overview of a multi-modal approach to assessing recovery following concussion in youth athletes. The described protocol uses pre- and post-concussion assessment of performance across a wide variety of domains and can inform the development of improved concussion rehabilitation protocols specific to the youth sport community.

Un Enfoque Multi-Modal de recuperación Evaluación de Atletas Jóvenes Siguiendo Concusión

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can ...

A Neuroscientific Approach to the Examination of Concussions in Student-Athletes

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement &#8220;may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an &#8216;impulsive&#8217; force transmitted to the head.&#8221; Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual&#8217;s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.

There is great variability in an individual&#8217;s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.

Un enfoque neurocientífica al Examen de conmociones cerebrales en estudiantes atletas

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Preparación y<em&gt; In Vitro</em&gt; Caracterización de Magnetized miR-modificado células endoteliales

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

Un método para cuantificar Rendimiento de las extremidades superiores en la Vida Diaria El uso de acelerómetros

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como<em&gt; In Vitro</em&gt; Herramienta de detección en la investigación de la audición

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Diseño de guías quirúrgicas CAD/CAM para la reconstrucción maxilar mediante un enfoque interno

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...