This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

El protocolo se describe a continuación sigue las directrices de UIUC comité de ética de la investigación humana. 1. Recogida y digestión de Tejidos quirúrgico Muestra <ol><li> Recoger la muestra de tejido del tumor después de la resección de colon derecho (Figura 1A y 1B). Recoger el tejido de un sitio sano adyacente también. </li><li> Transferir el tejido inmediatamente a un vial de 15 ml que contiene 12 ml de solución de HBSS. Conservar el vial en el hielo dentro de una caja de la espuma aislante. </li><li> Transporte el tejido que contiene vial a una campana de cultivo de tejidos para su posterior procesamiento en 45 min. Conservar el vial en un bloque de hielo en el interior de la campana. </li><li> Vierta el tejido suministrado en una placa de 6 pocillos que contiene 6-7 ml de solución de HBSS usando una pipeta. Nota: La cantidad de tejido por pozo no es crítica en esta etapa de aclarado. </li><li> Mantenga la placa de 6 pocillos en un bloque de hielo. Enjuague el tejido en solución HBSS dos veces. </li><li> Cortar el tejido limpiamente en secciones separadas con LIC estérilessor. Tejido Pique en secciones más pequeñas no mayor de 1-2 mm 3 en tamaño, con una hoja de bisturí estéril. </li><li> Pesar un vial tripsina 0,1% marcada (que contiene 1 ml de 0,1% de solución de tripsina) antes de transferir el tejido. Transferencia de ~ 20 mg de tejido en cada uno de los viales de 0,1% de tripsina con una pipeta. </li><li> Trate de minimizar la transferencia de HBSS en el vial tripsina para evitar una mayor dilución de tripsina. </li><li> Pesar los viales de tripsina después de la transferencia de tejidos; documentar la diferencia como la masa de tejido. </li><li> Verter ~ 20 mg de tejido y 1 ml de tripsina en cada pocillo de una placa de 24 pocillos. </li><li> Ponga la placa de 24 pocillos en un agitador en la nevera a 4 ° C durante 16-20 horas. Durante este período de espera o en un momento anterior, preparar sustratos de gel de poliacrilamida y funcionalizar con proteínas ECM como se describe en la sección 3. </li></ol> 2. La disociación enzimática de la muestra de tejido en suspensión individual de células y el cultivo de células aisladas en el ECM funcionalizado elástico Substrates y platos de poliestireno rígido <ol><li> Después de 16-20 horas de incubación en tripsina bien a 4 ° C en un agitador, transferir tejido de 4 ° C tripsina a 4 ° C vial medio de crecimiento completo (que contiene 2-3 ml de medio de crecimiento completo) usando una pipeta. Asegúrese de transferencia mínima de tripsina en medio de crecimiento. Nota: formulación completa medios de crecimiento es como sigue: RPMI 1640 medio base suplementado con suero de caballo a una concentración final de 10% y penicilina-estreptomicina al 1% de la solución total. </li><li> Coloque el tejido que contiene vial medios de comunicación en un baño de agua caliente a 37 ° C durante 12-15 min. </li><li> Transferir el tejido a un vial de 15 ml que contiene la solución HBSS usando una pipeta, agite suavemente. </li><li> Repita 2.3. </li><li> Retire el tejido de la solución HBSS y traslado al 0,1% solución de colagenasa (en HBSS) vial que contiene 1 ml de solución. Incubar durante 45 minutos a 37 ° C y 5% CO 2 ambiente en una incubadora de cultivo celular humidificado. Durante el período de espera, growt calienteh medios de comunicación en un vial de 15 ml a 37 ° C en baño de agua. </li><li> Saque todos los fragmentos de tejido en minimización pipeta colagenasa arrastre. Expulsar sólo los fragmentos de tejido en viales de medios de cultivo pre-calentado. </li><li> Colocar un filtro bien inserto de 40 micras en cada pocillo de una placa de 6 pocillos. Humedecer el filtro con 1 ml de medio de cultivo celular. </li><li> Se tritura el tejido en los medios de comunicación con una pipeta de vidrio de 10 ml varias veces hasta que el tejido está completamente disociado. </li><li> Mantener la punta de pipeta lo más cerca posible a la base del vial. </li><li> Depositar la solución sobre el filtro para eliminar los residuos y las partes no disociadas. </li><li> Centrifugar la suspensión celular se filtró en los medios de comunicación a 150 xg durante 5 min a TA. </li><li> Eliminar el sobrenadante y resuspender después sedimento celular con 2 ml de medio de cultivo fresco. </li><li> Contar las células utilizando un hemocitómetro (si es necesario). Aislado Seed células primarias en matriz extracelular (ECM) geles funcionalizados y platos de poliestireno en concentración deseada.</li></ol> 3. Preparación y funcionalización de diferentes Rigidez poliacrilamida (PA) Geles y poliestireno Sustratos Nota: Para la demostración visual de la Sección 3, los espectadores / lectores son referidos a un reciente Diario de experimentos visualizados (Jove) del artículo 17. <ol><li> La adopción de los protocolos publicados 18, 19, 12 mm activan 2 cubierta de cristal se desliza químicamente para asegurar la unión del hidrogel covalente. <ol><li> En primer lugar, la cubierta de cristal regalo desliza con 3- Aminopropyltrymethoxysilane (ATS) durante 7 minutos a temperatura ambiente. </li><li> Retire el ATS completamente con enjuague de agua DI y tratar cubierta se desliza con 0,5% glutaraldehído (diluido en PBS a partir de solución de glutaraldehído social 70%) solución durante 30 minutos. </li></ol></li><li> Obtener 2 soluciones de gel kPa mediante la mezcla de 5% w solución de acrilamida v / y 0,05% -methylenebisacrylamide solución de (bis) N &#39;en solución salina tamponada con HEPES 10 mM 20 N,. Utilice 8% w / v de acrilamida y 0,13% N, -methylenebisacrylamide solución (bis) N &#39;10 en solución salina tamponada con HEPES mM de gel de 10 kPa 20. <ol><li> En ambos casos, usar 1: 200 persulfato de amonio (10% w / v) y 1: 2.000 N, N, N &#39;, N&#39; -tetrametiletilendiamina (TEMED) como iniciador y catalizador para el proceso de polimerización, respectivamente. </li><li> Además, añadir 100 l perlas fluorescentes a 2 solución de gel kPa como marcadores fiduciarios 21, 22. </li></ol></li><li> Depositar una gota de 20 l de pre-polímero de solución de gel de PA en el 12 mm2 activado hoja de la cubierta de cristal. Coloque otra 12 mm 2 regulares hoja de la cubierta de cristal en la caída. Asegúrese de que la caída de los diferenciales entre los cubreobjetos por capilaridad. Invierta el sándwich para 2 geles kPa para asegurar que la mayoría de las perlas vienen cerca de la superficie de cultivo de células. </li><li> Curar el gel de PA durante 45 min a RT. </li><li> Despegar la hoja de la cubierta superior utilizando una sola máquina de afeitar borde. Nota: Durante el pelado, desprendimiento procede de uno de los bordesdel sándwich. El gel permanece adherida a la lámina de vidrio activado. </li><li> Almacene los geles en solución de PBS antes de su uso. </li><li> Funcionalizar geles y cristal con las moléculas de ECM, la fibronectina humana PA a una concentración de 50 g / ml métodos siguientes publicados 23. <ol><li> En pocas palabras, incubar sustratos con 2 ml puro hidrato de hidrazina O / N. </li><li> Retire hidrato de hidrazina y enjuague los sustratos a fondo con agua DI. </li><li> Lavar los sustratos con ácido acético al 5% durante 30 min. </li><li> Retire ácido acético y enjuague los sustratos a fondo con agua DI. </li><li> Mantenga los sustratos sumergidos en agua DI durante 30 min. </li><li> Incubar los sustratos con fibronectina oxidado durante 35 min a una concentración de 50 mg / ml. </li><li> Enjuague los sustratos con PBS en agitador a bajas rpm durante 10 min. </li><li> Incubar todos los sustratos a 37 ° C en 2-3 ml de medio de cultivo durante 30 minutos antes de la galjanoplastia de las células. Nota: las células de la placa en un poblar escasamented forma (1.000-3.000 células / cm 2). Cada hoja de vidrio gel cubierta necesita ser contenido en un 35 mm placa de Petri. Permitir que las células se adhieran completamente antes de cualquier microscopía (al menos O / N). </li></ol></li></ol> 4. Tracción microscopía de fuerza y ​​de inmunofluorescencia microscopía ensayos <ol><li> Ensayo de Tracción Microscopía de Fuerza <ol><li> Cálido tripsina-EDTA / solución de 0.25% 10% SDS a 37 ° C en un baño de agua durante 10 minutos antes de comenzar los experimentos de tracción. </li><li> Retire un gel de la incubadora de cultivo celular a la vez y colocar en la platina del microscopio fluorescente invertido (magnificación 32X). </li><li> Encuentra una sola célula en el campo de visión. Retire la tapa del plato de Petri. </li><li> Tome una imagen de contraste de fase de la célula (por ejemplo, la Figura 3). </li><li> Cambie el modo de imágenes de fluorescencia y seleccione el filtro apropiado. No mueva la platina del microscopio o de la muestra durante este tiempo. </li><li> Tome una imagen del displac perlas fluorescentescado por la tracción celular (Figura 4C). </li><li> Añadir 1 ml de tripsina / solución de SDS al plato de Petri para separar la célula de gel. No mueva la platina del microscopio o de la muestra durante este tiempo. Además, tomar una imagen de control de la célula para asegurar la eliminación completa de gel. </li><li> Tome una imagen de referencia (fuerza null) de las perlas después se retira de la célula. </li><li> Repita los pasos 4.1.2-4.1.8 para el resto de geles. </li><li> Hacer una pila de imágenes usando ImageJ de las dos imágenes obtenidas en el paso 4.1.6 y 4.1.8 para cada caso. Para generar la pila de imagen, utilice siguiente secuencia de comandos en ImageJ después de abrir las imágenes: Imagen → Pilas → Imágenes para apilar. </li><li> Para obtener el campo de desplazamiento y tracción, usar plugins ImageJ siguientes métodos publicados 21, 22 Obtener los códigos y tutoriales detallados para estos plugins utilizando el siguiente enlace:. <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/ima</a>gejplugins. <ol><li> Alinear las imágenes de la pila utilizando el plugin &#39;Matching plantilla&#39;. Uso siguiente secuencia de comandos en ImageJ después de abrir la pila imagen generada en el paso 4.1.10: Plugins → Matching Plantilla → Alinear Rebanadas de Pila → Aceptar. Guarde la pila de imágenes en consecuencia. Use esta nueva pila de imágenes en los siguientes pasos. </li><li> Obtenga el campo de desplazamiento mediante el PIV (partícula imagen velocimetría) plugin (Figura 4D). Uso siguiente secuencia de comandos después de abrir la pila imagen guardada en el paso 4.1.11.1: Plugins → → PIV iterativo PIV (Básico) → Aceptar → Aceptar este PIV y la salida → Ok. Guarde la salida de PIV en el mismo directorio que en la pila imagen original. Use esto como entrada en el siguiente paso. </li><li> Por último, el uso de FTTC (transformada de Fourier de tracción citometría) plugin para obtener el mapa de tracción (Figura 4E). Uso siguiente secuencia de comandos: Plugins → → FTTC Inspropiedades de los materiales ert, es decir, el módulo de Young y la relación entre PA gel → Aceptar → de Poisson Seleccione el archivo de salida PIV guardado en el paso 4.1.11.2 → Aceptar. Guardar FTTC resulta en el mismo directorio que en la pila de imagen y salida de PIV. </li></ol></li></ol></li><li> Inmunofluorescencia microscopía ensayos <ol><li> Tomar los sustratos a inmunotinción a la campana laminar y eliminar los medios de cultivo. </li><li> Enjuagar los sustratos con PBS y fijar las células con paraformaldehído al 4% en PBS durante 20 min a TA. </li><li> Lavar los sustratos con PBS durante 3 veces (5 min cada vez). En consecuencia, se incuban los sustratos con potenciador de señal de 500 l durante 30 minutos y enjuagar con PBS. </li><li> Se incuban las células con anticuerpo monoclonal anti-vinculina en una dilución 1: 250 en PBS durante 45 min a TA. Lavar los sustratos con PBS durante 3 veces (5 min cada vez). </li><li> Incubar las muestras con anticuerpo secundario Alexa Fluor 488 de IgG de cabra anti-ratón en una dilución 1: 200en PBS a temperatura ambiente durante 30 min. Lavar los sustratos con PBS durante 3 veces (5 min cada vez). </li><li> Para visualizar la estructura F-actina, incubar las células con TRITC faloidina conjugados a una concentración 50 mg / ml durante 45 min a RT. Lavar los sustratos con PBS durante 3 veces (5 min cada vez). </li><li> Imagen de las muestras mediante microscopio láser confocal de barrido (Figura 5A - 5D). </li></ol></li></ol>

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The authors have nothing to disclose.

La tensión de tracción celular ha surgido recientemente como un potencial indicador biofísico del estado metastásico 13. Sin embargo, no existen datos tracción experimental con células tumorales primarias en la literatura hasta la fecha. Además, el cultivo de las células del colon directamente aislados primaria en diferentes geles de poliacrilamida rigidez no se informa todavía. Por lo tanto, establecemos un colon primaria optimizadas las condiciones de cultivo de células en geles y poliestireno <st...

En los últimos años se ha convertido en cada vez más evidente que mecánica micro-entorno, además de los factores bio-químicas, juega un papel importante en la regulación de las funciones celulares. Las células pueden detectar y responder a la rigidez sustrato sobre el que se adhieren a (como en la cultura 2D) o rodeados por (como en cultivo 3D) 3-7. Al hacerlo, las células pueden modular su diferenciación 3, 4 morfología, migración / motilidad 5, las propiedades biofísicas 6, 7 crecimiento, y otros procesos. Las células cancerosas también responden a la rigidez de la matriz 2D y 3D utilizando una coordinada, combinación jerárquica mecano-químico de los receptores de adhesión y proteínas de la membrana de transducción de señales asociadas, la arquitectura citoesquelética, y motores moleculares 1, 2. Por ejemplo, las células epiteliales mamarias (MECs) formar parénquima normal acinar cuando se cultiva en sustratos 150 Pa que es similar a la rigidezde tejido mamario sano. Curiosamente, exhiben las características de un tumor en desarrollo, tanto estructurales como la transcripción, cuando se cultivan en sustratos rígidos (&gt; 5000 Pa) que imitan la rigidez de un estroma tumoral 8. Además, otro experimento muestra que la tumorigénesis de mama se acompaña de reticulación de colágeno y ECM de refuerzo 9. Experimentos recientes muestran que (HCT-8) células de carcinoma de colon humano muestran metástasis como fenotipo (MLP) cuando se cultivan en sustratos 2D tienen rigidez fisiológicamente relevante (20-47 kPa), pero no en muy rígido (3.6 GPa) sustratos 10- 12 .Estos células primero forman grupos de células tumorales y luego se disocian el uno del otro, a partir de la periferia. Como este epitelial a redondeadas morfológica (E a ​​R transición) se produce el cambio, proliferan, reducir célula-célula y célula-ECM adherencia, y convertirse migratoria. HCT-8 células cultivadas sobre sustratos de poliestireno muy duras, que no presentan estosrasgos malignos. Así, se ha planteado la hipótesis de que HCT-8 células se vuelven metastásico debido a su exposición a las micro-entorno adecuado. Vale la pena señalar que estos experimentos se llevan a cabo con las líneas celulares de cáncer inmortalizadas o células primarias de murino derivado, no con células cancerosas humanas primarias. Un estudio reciente propone que la tensión de tracción celular aumentada se puede utilizar como un potencial biofísico firma para las células metastásicas 13 .El estudio implica la medición de la fuerza de tracción para diferentes líneas celulares de cáncer humano en geles de poliacrilamida. Se encuentra que las células cancerosas metastásicas pueden ejercer significativamente mayor esfuerzo de tracción en comparación con las células no metastásicas en todos los casos 13. Sin embargo, estos resultados contradicen directamente los resultados publicados anteriormente en murino líneas celulares derivadas de cáncer de mama 14. Además, un estudio reciente pone de manifiesto diferencias notables entre las células humanas inmortalizadas y primaria en su pro remodelación del citoesqueletoperfiles de proteína y la supervivencia celular expresión de la proteína 15. Por lo tanto, es importante volver a visitar muchos de los ensayos biofísicos incluyendo tracción para las células cancerosas humanas primarias. Esto se abordará la cuestión de si las células primarias recapitulan inmortalizada líneas celulares de cáncer tendencia tracción. El protocolo descrito aquí está optimizado para el aislamiento de células primarias humanas de colon (tanto sanas y cancerosas), y para el cultivo de ellos en sustratos blandos (hidrogeles de poliacrilamida), así como en placas de Petri. El protocolo se basa en la digestión y la consiguiente disociación enzimática de muestra de tejido quirúrgico en suspensión de células individuales 16. Para nuestro conocimiento, esta es la primera demostración de cultivo aislado tumor de colon primario y las células normales directamente sobre sustratos de hidrogel blandas con microperlas fluorescentes embebidos para citometría de tracción. Sustratos gel transparente también permiten inmunotinción. Este ensayo reveló diferencias en la organización F-actina yadherencias focales en las células del colon humanas primarias como cambios de rigidez del sustrato. Esta plataforma de cultivo celular se abre la posibilidad de explorar diversas propiedades biofísicas de células humanas primarias tales como rigidez celular y la tracción como parámetros para pronósticos de cáncer.

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isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

Las células cancerosas responden a matriz de rigidez mecánica de una manera compleja usando un sistema coordinado, jerárquica mecano-químico compuesto de receptores de adhesión y proteínas de la membrana de transducción de señales asociadas, la arquitectura citoesquelética, y motores moleculares 1, 2. Mecanosensibilidad de las diferentes células cancerosas in vitro son investigado principalmente con las líneas celulares inmortalizadas o células primarias de murino derivado, no con células cancerosas humanas primarias. Por lo tanto, se sabe poco acerca de la mecanosensibilidad de células de cáncer de colon humanas primarias in vitro. Aquí, un protocolo optimizado se desarrolla que describe el aislamiento de las células del colon humanos primarios a partir de muestras de tejidos humanos quirúrgicos sanos y cancerosos. Las células del colon aislados son entonces cultivadas con éxito en sustratos funcionalizados por una matriz extracelular blando (2 kPa rigidez) y rígido (10 kPa rigidez) hidrogeles de poliacrilamida y poliestireno rígido (~ 3,6 GPa rigidez) (fibronectinaen este caso). Microperlas fluorescentes están incrustados en geles suaves cerca de la superficie de cultivo de células, y el ensayo de tracción se lleva a cabo para evaluar las tensiones contráctiles celulares utilizando software de acceso libre y gratuito. Además, la microscopía de inmunofluorescencia en diferentes sustratos de rigidez proporciona información útil sobre la morfología celular primaria, la organización del citoesqueleto y vinculina que contiene adhesiones focales como una función de la rigidez del sustrato.

El protocolo descrito anteriormente se emplea con éxito para múltiples muestras de tejido (n = 12) de cuatro pacientes diferentes según las directrices de la junta de revisión institucional. La figura 1A ilustra un tumor colorrectal representante de la derecha después de la cirugía de la que se obtienen los cortes de tejido para cultivos celulares. Una sección de tejido típico en solución HBSS después de la transferencia a la campana laminar para su posterior procesamiento se muestra en <stron...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Aislamiento de células tumorales de colon humanos primarios de tejidos quirúrgicos y cultivándolas Directamente en Elastic sustratos blandos de tracción Citometría

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.4K vistas.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

Video: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Rápido aislamiento de células tumorales viables de circulación de muestras de sangre del paciente

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Investigación

Bioingeniería

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Los tejidos de ingeniería músculo esquelético de células mioblastos murinos progenitoras y aplicación de estimulación eléctrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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 ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Patrones celular en fotolitográficamente Definido Parylene-C: SiO<sub&gt; 2</sub&gt; Sustratos

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Recuperación Quirúrgica, aislamiento y<em&gt; In vitro</em&gt; Ampliación del cruzado anterior humana derivados de las células del ligamento para aplicaciones de ingeniería de tejidos

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caracterización biomecánica de los tejidos blandos humanos utilizando sangría y Ensayo de tracción

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Aislamiento de células primarias de ganglio retiniano murino (RGC) por citometría de flujo

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Aislamiento de células madre mesenquimatosas del periostio Alveolar humano y efectos de la vitamina D en la actividad osteogénica de células derivadas del periostio

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Medición de la rigidez de sustratos de silicona blanda mecanobiología estudios utilizando un microscopio de fluorescencia de campo amplio

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.