Apreciamos profundamente Dr. Takeshi Ikeya y personal técnico en Toyo Gosei, Tokio, Japón para proporcionar placas de cultivo micropatterned termosensibles, así como el asesoramiento científico. También agradecemos a la Sra Satomi Ogura, Sra Sae Suzuki, la Sra Asuka Miyoshi y la Sra Katsue Morii para la asistencia técnica con los experimentos con animales. Este trabajo fue apoyado financieramente en parte por las JSP KAKENHI subvención-en-Ayudas a la Investigación Científica, el Centro del Programa de Innovación (COI) y el programa de innovación S- de la Agencia de Ciencia y Tecnología de Japón (JST), y las JSP Core- a-Core Programa, A. Redes Avanzadas de Investigación.

Se realizaron todos los estudios en animales con la aprobación del Comité de Cuidado de Animales y el Empleo de la Universidad de Tokio, Tokio, Japón. 1. Preparación de la célula <ol><li> Para hepatocitos primarios, seguir el protocolo para el aislamiento de hepatocitos de rata por un proceso de digestión con colagenasa de dos etapas modificado 13,14. <ol><li> Anestesiar Sprague Dawley (SD) ratas (macho, 5 semanas de edad) con anestesia por inhalación con isoflurano. Coloque una rata en una cámara conectada a una máquina de anestesia para proporcionar isoflurano para la cámara. Saque la rata después de quedarse dormido, y la puso sobre la mesa de operaciones con ventilación usando una máscara. Controlar el flujo de la isoflurano en aproximadamente desde 0,4 hasta 0,7 L / min mediante la comprobación de las condiciones de la rata. Perfundir los hígados de ratas Sprague Dawley (SD) ratas (macho, 5 semanas de edad) de la vena portal hepática con una solución especial compuesta de cloruro de sodio 8 g / L (NaCl), 400 mg / L de cloruro de potasio (KCl), 78 mg / Lde sodio dihidrato de fosfato de dihidrógeno (NaH 2 PO 4 · 2H 2 O), 151 mg / L de hidrógeno fosfato disódico dodecahidrato (Na 2 HPO 4 · 12H 2 O), 2,38 g / L 2- [4- (2-hidroxietil) -1 piperazinil] etanosulfónico (HEPES), 190 mg / L de ácido etilenglicol tetraacético (EGTA), 350 mg / L de carbonato de hidrógeno de sodio (NaHCO 3), y 900 mg / L de glucosa. </li><li> Circular solución de colagenasa a través del hígado. NOTA: La solución se compone de 500 mg / L colagenasa, / sal tamponada de L Hank 9,8 g, 2,38 g / ml HEPES, 556 mg / ml de hidrato de cloruro de calcio (CaCl 2 · H 2 O), 350 mg / L de NaHCO3, y el inhibidor de tripsina / L 50 mg, con el pH ajustado a 7,2. </li><li> Retire cuidadosamente el hígado, y picar suavemente sobre un plato usando una hoja de bisturí, añadir medio de Eagle modificado de Dulbecco (DMEM) suplementado con 10% de suero bovino fetal (FBS), y filtrar la suspensión celular a través de un 100 micrasde malla de nylon. Para eliminar cualquier desecho adicional, centrifugar la suspensión celular a 20 × g durante 1 min. En este paso, los hepatocitos son en el sobrenadante. </li><li> Repetir la etapa de centrifugación dos veces (un total de tres centrifugaciones). Finalmente, centrifugar los hepatocitos a 50 × g durante 3 min para recuperar ellos en la forma de un pellet. </li><li> Vuelva a suspender los hepatocitos a una concentración de 4 × 10 5 células / ml en un medio de cultivo especial compuesto por DMEM suplementado con 10% FBS, 1% de Pen-Strep-GLUT (PSQ), 1% de sulfóxido de dimetilo (DMSO), 0,1 mol / L de dexametasona, 0.5 mg / ml de insulina, 10 mmol / L de nicotinamida, 0,2 mmol / L ascorbato fosforilada (Asc-2P), y 10 ng / ml de factor de crecimiento epidérmico humano (hEGF) 15. Este medio especial es obligatorio para la preservación de la función hepática en condiciones in vitro. </li></ol></li><li> Para la obtención de MSC de rata, la eutanasia Sprague Dawley (SD) (varones, 5 semanas de edad) por la administración excesiva de isoflurane. Resecar el fémur y la tibia, y recoger las médulas óseas mediante la inserción de una aguja G 22 en el eje del hueso para tirar de la cadena a cabo con 10 ml de DMEM suplementado con 10% FBS. Recoger las células por filtración a través de una malla de nylon 100 micras. </li><li> Semilla las células en 10 platos cm de cultivo utilizando DMEM que contenía 10% de FBS y 1% de penicilina / estreptomicina. Para los experimentos de esferoide, utilizar las MSC dentro de 5 pasajes. </li></ol> 2. Preparación de esferoides de células 3D <ol><li> Comercialmente obtener placas de cultivo de micropatterned, en el que las zonas adhesivas celulares están dispuestos regularmente en 100 m de diámetro en una forma bidimensional, rodeado de zonas no adhesivas recubiertas por la matriz de PEG. NOTA: Para el trasplante de células, un recubrimiento adicional con el PIPAAm polímero termosensible es necesario para permitir el desprendimiento de células por el enfriamiento de las placas (véase el paso 5.1). Las fluctuaciones de temperatura pueden inducir cambios en la química de este polímero8-10. A 37 ° C, PIPAAm es ligeramente hidrófobo, permitiendo que las células a ser cultivadas bajo condiciones normales. Una disminución de la temperatura por debajo de 32 ° C resulta en una rápida hidratación del polímero, que conduce a la separación espontánea de las células. </li><li> Seed los hepatocitos o MSC en placas de 12 pocillos micropatterned a una densidad de 4 × 10 5 células / pocillo andincubate ellos a 37 ° C en un ambiente humidificado contiene 5% de CO 2. Las células se acumulan en las áreas de adhesión micropatterned y formar gradualmente esferoides redondas en 2 días. NOTA: Para la preparación de las células de control en un cultivo en monocapa, utilizar placas normales 12 pocillos y sembrar las células en una densidad idéntica, siguiendo un procedimiento similar al descrito anteriormente. </li></ol> 3. Preparación de Polyplex Nanomicelles <ol><li> Sintetizar un copolímero de bloques, PEG-pAsp (DET) (poli [N &#39;- [N - (2-aminoetil) -2-aminoetil] aspartamida]) (PEG Mw = 12,000, grado de polimerización (DP) de la PSAP (DET) segmento = 59), y un homopolímero que se compone de sólo el segmento catiónico [PSAP (DET)] (DP = 55), siguiendo los procedimientos descritos previamente por los autores 11, 16, 17. </li><li> Preparar una solución mixta del copolímero de bloque PEG-pAsp (DET) y el PSAP (DET) homopolímero con una relación molar igual de grupos amino residuales en 10 mM de tampón HEPES (pH 7,3) mediante el ajuste de la concentración de polímero de 33,3 mg / ml y 19,1 g / ml, respectivamente. NOTA: Las concentraciones de polímero descritas anteriormente son valores representativos para la preparación de nanomicelles con una relación molar residual de los grupos amino totales en los dos polímeros a los grupos fosfato en el (N relación A / P) pDNA de 10. La relación N / P puede variar dependiendo del tipo de célula y el propósito (para más detalles, véase la discusión). El uso combinado de los dos polímeros puede lograr tanto blindaje PEG efectiva y el funcionamiento de la PSAP (DET) para mejorar endosomalescapar (para más detalles, véase la discusión) 18. </li><li> Preparar la luciferasa Gaussia pDNA que codifica, GL4 luciferasa, o eritropoyetina por clonación del segmento de la expresión de los respectivos genes en el plásmido pCAG-GS (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html ) para obtener la expresión bajo el CAG promotor / potenciador utilizando un kit comercial de acuerdo con el protocolo del fabricante. Amplificar el pDNA en una cepa competente de Escherichia coli y purificar usando un sistema de purificación de ADN plásmido libre de endotoxinas. Determinar la concentración de pDNA a una absorbancia de 260 nm para obtener una solución / ml 150 mg en 10 mM de tampón HEPES (pH 7,3). </li><li> Para preparar los nanomicelles Polyplex, mezclar a fondo la solución pDNA (150 g / ml en 10 mM de tampón HEPES) y la solución premezclada de los dos polímeros en una proporción de 2: 1 (en volumen). </li></ol> 4. transfección de genes en esferoides <ol><li> Se incuban las células (hepatocitoso MSCs) durante 72 horas después de la siembra en las placas micropatterned para permitir la formación de esferoides maduros. Para la transfección génica, añadir 100 l de la solución nanomicelle Polyplex (que contienen 10 g de pDNA) a cada pocillo después de sustituir el medio de cultivo con 1 ml de medio fresco. Continuar la incubación con la solución nanomicelle durante 24 hrs. </li><li> Para un control usando un reactivo de transfección a base de lípidos, mezclar las soluciones de pDNA con el reactivo en una relación en peso de reactivo / pDNA de 3. Ajuste de la dosis final de pDNA a ser igual tanto para el reactivo a base de lípidos y métodos nanomicelle. </li></ol> 5. Recuperación y Trasplante de esferoides celulares <ol><li> Sustituir el medio de cultivo con 200 l de PBS frío y colocar las placas en hielo. NOTA: Por lo general, los esferoides pueden desprenderse en aproximadamente 15 minutos y pueden ser recuperados en forma de una suspensión para el trasplante. </li><li> Aspirar suavemente las células en el 200 lsuspensión con una jeringa con un 23 G 27 G o aguja para inyecciones in vivo. </li></ol> 6. Evaluación de la expresión transgénica <ol><li> Para la evaluación in vitro de la expresión de luciferasa Gaussia secretada en el medio de cultivo, recoger 50 - 100 l de medio de precisamente 24 hrs después de reemplazar con medio fresco. Estimar la expresión de luciferasa usando un sistema de ensayo de luciferasa de Renilla comercial y un luminómetro según el protocolo del fabricante. NOTA: La luciferasa Gaussia se mantiene estable en el medio de cultivo durante más de una semana. Por lo tanto, para evaluar la eficiencia en tiempo real de la expresión de los transgenes, sustituir el medio con uno fresco antes de recoger el medio de muestra. El momento del cambio de medio puede ser flexible. </li><li> Para la evaluación in vivo de la expresión del transgén en animales huésped después del trasplante de células, anestesiar BALB / c ratones nude (hembra; 7 semanasde edad) bajo anestesia por inhalación con isoflurano. <ol><li> Coloque un ratón en una cámara conectada a una máquina de anestesia para proporcionar isoflurano para la cámara. Saque el ratón después de quedarse dormido, y lo puso sobre la mesa de operaciones con ventilación usando una máscara. Controlar el flujo de la isoflurano aproximadamente a 0,2 hasta 0,5 L / min mediante la comprobación de las condiciones del ratón. </li><li> Inyectar 200 l de la suspensión celular que contiene esferoides transfectadas con el pDNA de luciferasa que expresan GL4 (como se describe en 4.1) en el tejido subcutáneo de la región abdominal. </li><li> Inmediatamente después de la inyección de D-luciferina (150 mg / kg; vía intravenosa), medir la expresión de luciferasa usando el sistema de formación de imágenes IVIS según el protocolo del fabricante. </li></ol></li><li> Para la evaluación de los efectos terapéuticos de la trasplante de células, inyectar 200 l de la suspensión celular que contiene esferoides transfectadas con el pDNA que codifica eritropoyetina en eltejido subcutáneo de la región abdominal. <ol><li> Recoger muestras de sangre por hemorragia submandibular obtener aproximadamente 200 l de sangre 19. Medir la hemoglobina y el hematocrito utilizando un analizador de muestra de sangre. NOTA: El número de células para el trasplante está regulada por el número sembradas en las placas. Desafortunadamente, es difícil determinar el número exacto de células porque el número dentro de esferoides no se puede medir. </li></ol></li><li> Después de los experimentos, coloque los ratones en una almohadilla eléctrica conectada con un controlador de temperatura hasta que el despertar de la anestesia. </li></ol>

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The authors declare that they have no competing financial interests.

En este protocolo, es fundamental para mantener la estructura 3D de esferoides durante las etapas de introducción de genes y la recuperación esferoide. Es esencial mantener una microambientes favorables para las células para evitar la muerte celular o la pérdida de la actividad celular. Por ejemplo, la secreción de albúmina, una función innata representante de los hepatocitos, está bien conservado en los esferoides de hepatocitos, mientras que los hepatocitos en el cultivo monocapa convencional pierden rápidame...

Terapia de trasplante de células ha atraído una gran atención para el tratamiento de diversas enfermedades intratables. La actividad y la vida media de los factores bioactivos que son secretadas por las células trasplantadas son esenciales para mejorar la eficacia terapéutica de un sistema de trasplante de células. La modificación genética de las células antes del trasplante es una técnica beneficiosa para regular y manipular las funciones celulares, incluyendo la secreción de los factores bioactivos. También es importante mantener un microambiente favorable para las células para evitar la muerte celular o pérdida de la actividad celular. Tridimensional de cultivo (3D) de células esferoide, en el que las interacciones de célula a célula están bien conservados, es prometedor para este propósito, por ejemplo, para mejorar la secreción de albúmina de los hepatocitos primarios y promoción de la diferenciación multi-linaje de células madre mesenquimales (MSCs ) 1-7. En este estudio, un nuevo sistema de combinación de spheroid cultura y la transfección de genes se utiliza para servir como una plataforma para el trasplante de células modificadas genéticamente. Para la creación de células esferoides, se utiliza un sistema de cultivo en placas de cultivo esferoide micropatterned. En estas placas, las áreas de adhesión celular de 100 m de diámetro están dispuestos regularmente en una forma de dos dimensiones y están rodeadas por áreas no adhesivas recubiertas por una matriz 3 PEG. Por la siembra de un número adecuado de células, se forman matrices de esferoides en 3D de 100 micras de diámetro correspondiente al lecho de cultivo microestructurada. Los esferoides se recuperan sin interrumpir su estructura 3D mediante el uso de placas de cultivo celular termosensibles, que se revistieron con un polímero termosensible, poli (iso-propilacrilamida) (PIPAAm) 8-10. La arquitectura micropatterned se construye sobre las placas termosensibles (a medida construido). Por simplemente bajando la temperatura de las placas, los esferoides se separan de la cama de cultivo y se dispersand en tampón fosfato salino (PBS). Por lo tanto, un gran número de esferoides con un tamaño uniforme de 100 micras se puede obtener en la forma de una suspensión inyectable. <img alt="Figura 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Figura 1. Representación esquemática del sistema de cultivo esferoide en una placa microestructurada. La modificación genética se consigue mediante transfección de genes usando la no viral portador del gen original, nanomicelle Polyplex. Se compone de ADN de plásmido (pDNA) y polietilenglicol (PEG) copolímeros de bloque -polycation 11. Estos tienen una estructura de núcleo-corteza característica que consiste en una cáscara de PEG y un núcleo interno de pDNA condensada, lo seguro y eficaz introducción de genes en células con fines terapéuticos 11. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de thi</a>s figura. <img alt="Figura 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> Figura 2. Estructura de la nanomicelle Polyplex formado por el complejo de ácidos nucleicos y copolímeros de bloque de PEG-bloque-policatión. En este estudio, la principal ventaja de esta técnica es que la estructura de esferoide no se interrumpe durante transfección de genes por los nanomicelles. Después de transfecciones nanomicelle mediada de rata esferoides de hepatocitos primarios, prolongada expresión del transgen se obtiene durante más de un mes con la secreción de albúmina continua de los hepatocitos en un nivel comparable a la de esferoides no transfectadas 12. La expresión del transgén y la secreción de albúmina a partir de los esferoides también se mantienen después de la recuperación de las placas termosensibles. Es evidente que nanomicelles pueden facilitar de manera segura la introducción de genes sin perjudicar las funciones innatas de la hepatocytes. Por lo tanto, la combinación de células esferoides cultivadas en placas micropatterned termosensibles con la introducción de genes usando nanomicelles es una plataforma prometedora para el trasplante de células modificadas genéticamente. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

Para mejorar la eficacia terapéutica del trasplante de células, se desarrolló un sistema de trasplante de genéticamente modificados, esferoides inyectables. Los esferoides de células se preparan en un sistema de cultivo en placas de micropatterned recubiertas con un polímero termosensible. Un número de esferoides están formados en las placas, correspondientes a las áreas de adhesión celular de 100 m de diámetro que están dispuestos regularmente en una forma bidimensional, rodeadas por áreas no adhesivas que están recubiertas por un glicol de polietileno (PEG) matriz. Los esferoides se pueden recuperar fácilmente como una suspensión líquida mediante la reducción de la temperatura de las placas, y su estructura está bien mantenido pasándolos a través de agujas de inyección con un calibre lo suficientemente grande (más de 27 G). La modificación genética se consigue mediante transfección de genes usando la no viral portador del gen original, nanomicelle Polyplex, que es capaz de introducir genes en las células sin interrumpir la estructura esferoide. Para primesferoides de hepatocitos ary transfectadas con un gen de luciferasa que expresan, la luciferasa se obtiene de forma sostenible en animales trasplantados, junto con la función de los hepatocitos conservados, como se indica por la expresión de albúmina. Este sistema se puede aplicar a una variedad de tipos de células incluyendo células madre mesenquimales.

Transfección de genes de la luciferasa que expresan pDNA Gaussia se realizó en los esferoides formados por los hepatocitos o MSCs utilizando nanomicelles Polyplex o el reactivo de transfección basado en lípidos de control 12. Los nanomicelles inducidos casi ningún cambio en la estructura esferoide en comparación con esferoides no transfectadas en las placas micropatterned, mientras que el reactivo de control interrumpe significativamente la estructura de un día después de la transfección (Fig...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

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

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

La transfección de genes hacia células Esferoide sobre placas micropatterned Cultura para el trasplante de células genéticamente modificadas

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

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K vistas.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Superficies micropatterned para estudiar las interacciones de ácido hialurónico con células de cáncer

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Investigación

Bioingeniería

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Una mejor visualización y análisis cuantitativo de los efectos de drogas Uso de células micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabricación de hidrogeles micropatterned para sistemas de cultivo neuronal usando fotolitografía dinámica de proyección Máscara

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotación de sistemas de cultivos celulares de cultivo de células humanas: células humanas trofoblásticas como un modelo de

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Citometría de masas: Protocolo para la afinación y servicio diario muestras de células en un citómetro de masas CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Trasplante De InducidaS Pluripotentes Derivadas De Células Madre Mesoangioblastos Como Progenitores Miógenos En Modelos De Ratón De La Regeneración Muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

El estiramiento células micropatterned en una membrana de PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Microfluidic genipina Deposición Técnica de Cultura extendido de micropatterned vasculares musculares Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentación Del Crecimiento De Las Células Endoteliales En Placas De 6 Pozos En Una Coctelera Orbital Para Estudios Mecanobiológicos

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Un método de cultivo para mantener las células madre hematopoyéticas humanas quiescentes

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...