We thank Simon Hayward (Vanderbilt University Medical Center) for the BPH-1 cells; and Thomas Cox and Janine Erler (Biotech Research &amp; Innovation Centre, University of Copenhagen) for their assistance with rheological measurements. MR-T was funded by Worldwide Cancer Research, formerly The Association of International Cancer Research (Grant 08-0803 to JS), The British Embassy Montevideo and Agencia Nacional de Investigacion e Innovacion (UK_RH_2015_1_2 to MR-T). MC was supported by Prostate Cancer UK (Grant S14-017 to JS and GS). KW was funded by The China Scholarship Council. MAM was funded by The Saudi Arabian Cultural Bureau.

<ol>
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The authors have nothing to disclose.

Un protocolo para la generación de 3D ​​acinos glandulares de MECs en geles de RBM puros 6 fue modificada en un estudio previo de la adición de 4 mg tipo / ml de colágeno I a la matriz de la GBR. La adición de colágeno resultó en el módulo de elasticidad del gel RBM pasando de 175 ± 37 a 1589 ± 380 Pascales. Este aumento de 9,1 veces en la rigidez modula el crecimiento, la supervivencia, migración y diferenciación de MEC 21. El protocolo fue modificado de nuevo mediante la inclusión de una etapa de tratamiento con D - (-) - ribosa para promover la reticulación no enzimática del colágeno tipo I que se había añadido al gel de la GBR. Se encontró que el aumento de 15 veces resultante en la rigidez a cooperar con la transformación oncogénica de MECs para promover su comportamiento invasivo 22. El enfoque experimental de tipo adición de colágeno I a geles RBM facilita la interacción directa de MECs con fibras de colágeno, que sólo se produce en el tejido humano después de la barrera física entre el estromay el epitelio proporcionada por el BM se somete a la degradación proteolítica. Mediante la generación de los acinos glandulares 3D a partir de los PEC en geles de RBM puros pre-tratados con GLA, el protocolo actual abre el camino para estudiar cómo BM rigidez per se puede desencadenar su comportamiento invasivo (Figura 3). Los niveles de rigidez BM inducidas en este protocolo tienen relevancia fisiológica. La incubación con GLA 50 mM durante 6 hr y 14 hr aumentó, respectivamente, los módulos de elasticidad del gel RBM puro a 175 ± 90 y 322 ± 160 en comparación con 122 ± 55 Pascales en geles de RBM tratados con PBS (Tabla 1). Este aumento de 1,7 a 3,2 en la GBR rigidez recapitula el aumento de 2,5 a 3,4 veces en la rigidez observada en comparación con maligna de la próstata normal o tejido de BPH 23-26. Como se describe en una publicación reciente 13 los cambios morfológicos inducidos por la acumulación de AGE y la rigidez de gestión por resultados en el PEC acinos se puede cuantificar por un cambio estadísticamente significativo del arundada de forma poligonal, disminución de células que migran desde la que sobresalen en el acina-AGE rica RBM (Figura 3) luminal / área total de los acinos e. Inmunotransferencia también se puede utilizar para evaluar los marcadores de EMT (por ejemplo, pérdida de E-cadherina 13) y el comportamiento contráctil (por ejemplo ligera de la miosina fosforilada cadena-2, pMLC2 13) en los PEC cultivadas en normal frente rígida RBM (Figura 3). La evaluación adicional utilizando la tinción de inmunofluorescencia y microscopía confocal se puede aplicar a visualizar el BM (por ejemplo, laminina, colágeno IV y la acumulación de AGE 13), celular polaridad apical a-basal (por ejemplo, la localización apical de EEA1: antígeno endosomal temprano 1; y GM130: 130 kDa marcador de Golgi cis-13) y los patrones celulares de moléculas de adhesión (por ejemplo, e-cadherina localización de uniones célula-célula 13) (Figura 3). <img alt = &quot;Figura 3&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig3.jpg&quot; /&gt; Figura 3:. Visión general de los diferentes protocolos que aquí se presenta el diagrama describe cómo preparar y endurecer la membrana basal reconstituida (RBM) con glicolaldehıdo (reacción de Maillard), la forma de sembrar las células a la rigidez de gestión por resultados, la forma de analizar la rigidez RBM ( extensión de la reacción de Maillard) y los procedimientos que se pueden utilizar para analizar los cambios celulares y moleculares inducidos por la GBR-AGE rico. AGE, productos finales de glicación avanzada; BM, la membrana basal; DAPI, 4 &#39;, 6-diamidino-2-fenilindol; EEA1, antígeno temprano endosomal 1; GAPDH, gliceraldehído-3-fosfato deshidrogenasa; GLA, glicolaldehıdo; GEE, éster etílico de glicina; GM130, 130 kDa marcadores cis-Golgi; p-MLC2 (Thr18 / Ser19), miosina de cadena ligera 2 fosforilada en sitios de treonina y serina 18 19; RBM, membrana basal reconstituida; SDS-PAGE, dodecil sulfato de electroforesis en gel de poliacrilamida de sodio. Para la barra RWPE1 acinos de escala = 10 micras; para las células tumorales PC3Barra de escala = 100 micras esferoides. Esta cifra ha sido modificado de referencia 13. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> pasos para solucionar problemas serán necesarias si D - (-) - ribosa es elegido como el agente de reticulación de la GBR. Durante el desarrollo del protocolo se encontró que el tratamiento con 1 M D - (-) - ribosa durante 72 horas, como se describe anteriormente para los geles de la GBR / colágeno 22, dio lugar a la deshidratación y la contracción de los geles de la GBR. La evaluación de las concentraciones más bajas de D - (-) - ribosa y los tiempos de tratamiento más cortos puede ayudar a superar esta limitación. Una limitación potencial en futuras aplicaciones del protocolo se podría encontrar en aplicaciones que requieren niveles más altos de la GBR rigidez. Si se utilizan tiempos de incubación más largos y concentraciones más altas de GLA para inducir niveles más altos de la GBR rigidez gel será necesario<html> evaluar si estas condiciones de tratamiento tienen un impacto sobre la supervivencia y la proliferación celular, como se ha descrito previamente 13. También hay que señalar que la incubación de células RWPE-1 con suero induce una transición EMT-como fenotípica y la exposición a suero o materiales que contienen suero debe ser evitado. Por ejemplo, si los experimentos implican la transfección de interferir corta de ARN oligonucleótidos (siRNA), el procedimiento debe ser optimizado utilizando células RWPE-1 cultivadas en KSFM, sin cambiar las células a los medios bajo transfección suero. Este inconveniente podría comprometer el nivel de silenciamiento génico logra al utilizar siRNA transitoria se acerca en el modelo. Para algunas dianas proteicas se le recomienda emplear shRNA vectores inducibles para el silenciamiento de genes sintonizable y la disminución en los niveles de proteína deseada. Las adaptaciones que incorporan reticulación enzimática por células estromales o células tumorales lisil oxidasa asociada (LOX) 17 también se podrían incorporar en los modelos futuros. jove_content &quot;&gt; Este protocolo facilitará el estudio futuro de los mecanismos pro-invasivas provocadas por la edad que dependen rigidez BM en los PEC (RWPE-1, HPB-1) y la evaluación de los objetivos anti-metastásicos en los PTC invasivos (PC3). Teniendo en cuenta que la HPB se considera que es un trastorno metabólico 27, este protocolo también allana el camino hacia nuestra mejor comprensión de la relación entre los trastornos metabólicos y aumento del riesgo de cáncer de próstata. Teniendo en cuenta que la rigidez BM inducida por su exposición a los AGE puede ser un desencadenante de invasividad en otro tipo de cáncer tipos, que serán de interés para utilizar el protocolo para establecer modelos similares que incorporan células epiteliales normales y células tumorales de otros órganos (por ejemplo, mama, colon, ovarios, páncreas). Los pasos críticos en el protocolo, junto con sus tiempos, se resumen en la Figura 4. Durante la etapa inicial, es esencial para mantener la solución madre de la GBR a 4 ° C, mientras que se descongele para evitar su polymerization. Puntas de pipeta no se deben colocar en la solución de stock rM hasta que se hayan enfriado a 4 ° C. Para el siguiente paso también es importante para asegurar que los portaobjetos de cámara se hayan equilibrado a 4 ° C antes de que se recubren con la solución de la GBR. Tan pronto como la temperatura de la solución se incrementa por encima de la GBR 4 ° C que sufrirá polimerización irreversible para formar un gel. Es esencial que la gestión por resultados no se altera durante la etapa de polimerización para asegurar que forma una superficie plana adecuada para el cultivo de células y análisis microscópico. La duración de la incubación con GLA con o sin inhibidores de la reacción Maillard (cianoborohidruro de sodio y amingoguanidine) determinará la rigidez del gel se convierte en la GBR. Se recomienda el uso de una incubación de 6 h con GLA si se requieren condiciones semi-rígidos, y la incubación 14 h si se requieren condiciones severas (Tabla 1). tiempos de incubación alternativos o concentraciones de GLA se pueden utilizar si diferentes nivelesde la rigidez de los deseados. En este caso el análisis reológico de los geles de RBM ser necesario incorporar como un paso esencial. Después de la etapa de enfriamiento rápido de la reacción de Maillard por incubación con GEE y las etapas de lavado subsiguientes con PBS, los geles RBM se pueden utilizar inmediatamente o se almacenaron a 4 ° C durante un máximo de 48 horas antes de su uso para el cultivo celular. Una vez los cultivos celulares se establecen es importante para cambiar el medio de cultivo (incluyendo cualquier tratamientos) cada dos días. Se recomienda mantener los cultivos de células 3D por 3-12 días de acuerdo con los parámetros objeto de investigación. Para 3D PEC acinos se recomienda analizar los cultivos después de 6 días, y para 3D de PTC esferoides análisis se recomienda después de 3 días de cultivo en la primera instancia. <img alt="Figura 4" src="/files/ftp_upload/54230/54230fig4.jpg" /> Figura 4:. Descripción simple del Protocolo con etapas críticas y sincronizaciones indicado la flow diagrama representa cómo preparar y endurecer la membrana basal reconstituida (RBM) con glicolaldehıdo (reacción de Maillard) con pasos críticos y los tiempos indicados. Puntos en los que el protocolo se puede detener, y geles RBM almacena, también se indican. RBM, membrana basal reconstituida; GLA, glicolaldehıdo; GEE, éster etílico de glicina; ON, durante la noche; PBS, solución salina tamponada con fosfato; RT, la temperatura ambiente. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

The basement membrane (BM) is a sheet of specialized extracellular matrix (ECM) that maintains stable tissue borders by separating layers of epithelial cells from the stroma1. Covalent crosslinking between adjacent triple helices of collagen IV in the BM stabilizes their lateral association by establishing an irregular network of super-twisted helices2. These collagen IV lattices act as a scaffold for its interaction with laminin and other BM components1. The structural arrangement of the BM provides it with the mechanical strength and rigidity necessary for the normal development of glandular epithelia3. 
During aging and disease the BM progressively thickens and stiffens3,4. For example, a 3-fold increase in the elastic modulus (E) of the ocular BM occurs between the ages of 50 and 80 in the normal population, and this stiffening is further exacerbated in metabolic disorders like diabetes5. The structural and biomechanical changes in the BM that result in its increased stiffness occur when its ECM components, collagen IV and laminin, become non-enzymatically crosslinked following their exposure to advanced glycation endproducts (AGEs).
The purpose of the method described here was to establish a model for the investigation of how BM stiffness, due to AGE exposure, promotes prostate epithelial cell (PEC) and prostate tumour cell (PTC) invasiveness in the context of the switch to metastatic prostate cancer (PCa). To do this a previous method used for generating 3D glandular acini from mammary epithelial cells (MECs) in reconstituted rBM gels6 was adapted to include an additional step where the rBM gels are pre-treated with glycolaldehyde (GLA). Several techniques for assessing GLA induced crosslinking and stiffening of pre-treated rBM gels are described, including photometric analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), confocal microscopy and rheometric analysis. The prostate cell types selected for culture on the pre-stiffened rBM include: RWPE-1, PECs derived from a normal prostate gland7; BPH-1, PECs derived from a prostate gland affected by BPH8; and PC3, metastatic PTCs derived from a secondary tumor located in the vertebral bone of a prostate cancer (PCa) patient9. 
In addition to advancing the study of prostate gland pathology, the protocol for stiffening of rBM gels by their treatment with GLA can be adapted to investigate how BM stiffness contributes to other age-related pathologies and metabolic disorders. For example, the model can be directly applied to investigate how metastatic cancer is induced by BM stiffness in organs such as the breast, colon, ovary and pancreas by the incorporation of appropriate cell types. Furthermore, the protocol can be adapted to investigate how stiff BM promotes biomechanical mechanisms of disease progression in diabetes-related microvascular disease, retinopathy, nephropathy and neuropathy.

<table><tbody><tr><td>&lt;strong&gt;I - Material for monolayer culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BPH-1</td><td>CaP Cell Line Database</td><td>PCaCL-132</td><td>Contact: simon.hayward@mcmail.vanderbilt.edu</td></tr><tr><td>Complete keratinocyte serum-free media&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>17005-075</td><td>Do not warm at 37 &amp;ordm;C before use</td></tr><tr><td>Fetal calf serum&amp;nbsp;</td><td>First Link UK Ltd</td><td>02-00-850</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>PC3&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-1435</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate Buffer Saline (Dulbecco A) tablets</td><td>Oxoid</td><td>BR0014G</td><td></td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R5886</td><td>warm in 37 &amp;ordm;C water bath before use</td></tr><tr><td>RWPE-1&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-11609</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;II - Material for 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>Aminoguanidine hydrochloride</td><td>Sigma-Aldrich</td><td>396494</td><td>Irritating to eyes, respiratory system and skin</td></tr><tr><td>Chamber slides, 8-well&amp;nbsp;</td><td>Thermo Scientific Nunc Lab-Tek</td><td>TKT-210-816M</td><td></td></tr><tr><td>Culture Matrix reconstituted basement membrane (rBM) reduced growth factor extract&amp;nbsp;</td><td>AMS Biotechnology</td><td>3445-005-01</td><td>Store Basement membrane (BM) at -80 &amp;ordm;C in aliquots</td></tr><tr><td>Cyanogen bromide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C91492</td><td>Toxic by contact skin and inhalation</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>695076</td><td></td></tr><tr><td>Glycine ethyl ester hydrochloride (GEE)</td><td>Sigma-Aldrich</td><td>50060</td><td>Irritating to eyes</td></tr><tr><td>Glycolaldehyde dimer (GLA)</td><td>Sigma-Aldrich</td><td>G6805</td><td></td></tr><tr><td>Sodium cyanoborohydride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71435</td><td>Highly flammable; Toxic by contact skin and inhalation</td></tr><tr><td>Syringe filter 0.22 microns</td><td>Appleton Woods</td><td>BC680</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;III - Material to quantify Maillard reaction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>ThemoFisher Scientific</td><td>D3571</td><td>light sensitive and store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Cloning cylinders</td><td>Sigma-Aldrich</td><td>C1059</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11001</td><td>light sensitive</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11034</td><td>light sensitive</td></tr><tr><td>Goat serum</td><td>Abcam</td><td>ab7481</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Vectashield mounting media&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Mouse anti-pentosidine clone PEN-12 mAb</td><td>TransGenic Inc</td><td>KH012</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>F8775</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-human collagen IV polyclonal antibody&amp;nbsp;&amp;nbsp;</td><td>Acris Antibodies</td><td>R1041</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-laminin A/C pAb</td><td>Santa Cruz Biotechnology Inc</td><td>sc-7292</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton-X100)</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate (Tween-20)</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Dialysis cassette Slide-A-Lyzer</td><td>ThemoFisher Scientific</td><td>66333</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;IV - Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ARG2 controlled strain rotational rheometer</td><td>T.A. Instruments</td><td></td><td></td></tr><tr><td>Axiovert S100 (20X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CO2 controlled humidified incubation chamber for Zeiss Axio S100 microscope</td><td>Solent Scientific</td><td></td><td></td></tr><tr><td>Confocal Axiovert 200M (40X, 63X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Olympus LH50A microscope fitted with a digital camera using phase-contrast&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>PHERAstar Plus plate reader spectrophotometer</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;V - Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image J 1.47v</td><td>National Institute of Health, USA</td><td></td><td></td></tr><tr><td>MetaXpress</td><td>Molecular Divices</td><td></td><td></td></tr></tbody></table>

how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Aquí se describe un protocolo que se puede utilizar para estudiar el microambiente biofísica relacionada con un aumento del grosor y la rigidez de la membrana basal (BM) durante patologías relacionadas con la edad y trastornos metabólicos (por ejemplo, cáncer, diabetes, enfermedad microvascular, retinopatía, nefropatía y neuropatía) . La premisa de este modelo es la reticulación no enzimática de la matriz BM reconstituido (GBR) por tratamiento con glicolaldehıdo (GLA) para promover el producto final de glicación avanzada generación (AGE) a través de la reacción de Maillard. Ejemplos de técnicas de laboratorio que se pueden utilizar para confirmar la generación de AGE, la reticulación no enzimática y mayor rigidez en GLA tratados RBM se describen. Estos incluyen la preparación de la GBR nativa (tratado con solución salina tamponada con fosfato, PBS) y rígido RBM (tratados con GLA) para la determinación de: su contenido de AGE por análisis fotométrico y microscopía de inmunofluorescencia, su no-enzimática por reticulación de sodio poliacrilamida dodecilsulfatoelectroforesis en gel (SDS PAGE), así como la microscopía confocal, y su aumento de la rigidez usando reometría. El procedimiento descrito aquí se puede utilizar para aumentar la rigidez (módulos de elasticidad, E) de la GBR hasta 3,2 veces, de acuerdo con las mediciones realizadas en sanos frente a tejido de la próstata humano enfermo. Para recrear el microambiente biofísica asociada con el envejecimiento y la glándula próstata enferma tres tipos de células de la próstata se introdujeron en la GBR nativa y rígido RBM: RWPE-1, las células epiteliales de la próstata (PEC) derivada de una glándula de la próstata normal; BPH-1, los PEC derivados de una glándula de la próstata afectados por hiperplasia prostática benigna (BPH); y PC3, células metastásicas derivan de un tumor óseo secundario procedente de cáncer de próstata. Múltiples parámetros pueden ser medidos, incluyendo el tamaño, forma y características invasoras de los acinos glandulares 3D formado por RWPE-1 y la HBP-1 sobre los nativos frente rígida RBM, y la longitud de celda promedio, la velocidad migratoria y la persistencia del movimiento de las células de spher 3Doids formados por células PC3 en las mismas condiciones. vías y la localización subcelular de las proteínas de señalización celular también se puede evaluar.

3D de próstata cultivadas en Acini rígido RBM Después de 6 días de cultivo, los PEC derivadas de tejido normal de la próstata (RWPE-1) (Figura 1A) y tejido de BPH (HPB-1) (Figura 1B) forma acinos en nativo (tratados con PBS) RBM que se organiza en esferoides uniformes de epitelio Células. Estos acinos también tienen las características de los PEC altamente organizados con el espacio-apical-basal de polaridad y una luminal visibles 13,20. El acinos formado por los PEC derivados de tejido de la próstata normal (RWPE-1) (Figura 1A) y el tejido BPH (BPH-1) (Figura 1B) en rigidizadas RBM (-AGE rico) (tratado con GLA) tener una arquitectura interrumpido (desplazando desde esferoidal de forma poligonal y las células que sobresale / migración de los acinos a la era rica en RBM) (Figura 1A). Estas<html> acinos se caracterizan también por los PEC altamente desorganizado que han perdido su polaridad apical-to basal con una pequeña o inexistente espacio luminal 13. <img alt="Figura 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> Figura 1:. Próstata células epiteliales cultivadas como 3D glandular Acini en la membrana reconstituido sótano nativo y rígido (GBR) (A) Imágenes de campo claro de células RWPE-1 se cultivaron durante 12 horas hasta 6 días en geles de RBM tratados con PBS (nativo) o glicolaldehıdo 50 mM durante 14 horas (AGE-rica; rígida); Barra de escala = 50 micras. (B) células de HPB-1, que se cultiva como se describe en el panel A; Barras de escala = 50 m; los datos son representativos de 3 experimentos independientes. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <html> Tabla 1 &quot;src =&quot; / files / ftp_upload / 54230 / 54230table1.jpg &quot;/&gt; Tabla 1:. Características de próstata epitelial RWPE-1 acinos Cultivado en las Nativo, semi-rígido y rígido membrana reconstituido sótano (RBM) RWPE-1 acinos se cultivaron en RBM pre-tratados con PBS durante 14 horas (nativo), glicolaldehıdo (GLA ) durante 6 horas (semi-rígido) o GLA durante 14 horas (rígida). Por forma acinar, el porcentaje (%) ± desviación estándar (SD) de vuelta, acinos semi-poligonal y poligonal se calcularon a partir de 5 experimentos independientes (50 acinos cuantificaron por condición). acinar tamaño relativo se calculó (RBM nativa = 100%) de 3 experimentos independientes. Para invasividad,% ± SD acinos con una o más células que sobresalen se calcula a partir de 3 experimentos independientes. factor de cambio se calcula dividiendo el valor medio obtenido en condiciones semi-rígidas o rígidas por el valor correspondiente para condiciones nativas. Los valores de p calculados utilizando la prueba t de Student (α = 0,05). ontenido &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; dependen de la edad mayor rigidez RBM promueve la migración de células tumorales de próstata PC3 Células PC3 cultivadas en RBM nativa migran al mantener contacto célula-célula continua, mientras que las células PC3 cultivadas en AGE-rica (rígida) RBM se mueven independientemente uno del otro (Figura 2A). Después de 72 horas en cultivo de células PC3 formar focos (esferoides) el nativo (tratados con PBS) GBR, mientras que las células PC3 sobre rígida RBM (EDAD-rica) No de esferoides y migrar de forma independiente (Figura 2B). Células PC3 sobre la RBM (EDAD-rica) rígidas son más alargada que las células PC3 cultivadas en RBM nativa (Figura 2C). En células PC3 rígida RBM migran más rápidamente que las células PC3 cultivadas en RBM nativa (Figura 2D). En células PC3 rígida RBM muestran una disminución de la persistencia en comparación con las células PC3 cultivadas en RBM nativa (Figura 2E). <img alt = &quot;Figura 2&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig2.jpg&quot; /&gt; Figura 2:. La migración de próstata de células tumorales en la membrana reconstituido sótano nativo y rígido (GBR) (A) Imágenes de campo claro de células PC3 cultivadas en geles de RBM tratados con PBS (nativo) o glicolaldehıdo 50 mM durante 14 horas (AGE-rica, rígido) . Las células fueron imágenes utilizando un microscopio de campo claro (objetivo 10X) y una tasa de adquisición de imagen 1 por hora durante 12 horas seguido de la célula de seguimiento para generar trayectorias. Imágenes mostrados corresponden a los puntos de tiempo después de 0, 3, 6, 9 y 12 hr. Trayectorias de células individuales se muestran para el punto de tiempo 12 horas. Barra de escala = 100 micras. Células (B) PC3 cultivadas en RBM nativa o rígido durante 72 horas, y la imagen como se describe en el panel (A). Barra de escala = 100 micras. Detalle muestra el área asignada a la ampliación de 2x. (C) Media ± SD longitud de la célula (m); diferencia significativa entre la GBR nativa y rígido RBM (p = 1,2 x 10 -23). ( <strong&gt; D) Media ± SD de velocidad (m / h) calculada a partir de las trayectorias de células; diferencia significativa entre la GBR nativa y rígido RBM (p = 0,004). (E) Media ± SD persistencia del movimiento de las células (relación D / T, donde D = distancia desde el principio hasta el final de la trayectoria celular, T = longitud total de la trayectoria celular); diferencia significativa entre la GBR nativa y rígido RBM (p = 0,0007). Para los paneles se analizaron CE&gt; 10 células, los datos son representativos de 3 experimentos independientes. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Aquí se explica un protocolo para modelar el microambiente biofísico donde la reticulación y el aumento de la rigidez de la membrana basal (BM) inducida por productos finales de glicación avanzada (AGEs) tiene relevancia patológica.

Como estudiar membrana basal para la rigidez como un disparador de Biofísica en el cáncer de próstata y otras patologías relacionadas con la edad o enfermedades metabólicas

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

how-to-study-basement-membrane-stiffness-as-biophysical-trigger

Research

JoVE Journal

Cancer Research

9.6K vistas.  Universidad de la República (UDELAR).  Aquí se explica un protocolo para modelar el microambiente biofísico donde la reticulación y el aumento de la rigidez de la membrana basal (BM) inducida por productos finales de glicación avanzada (AGEs) tiene relevancia patológica. 

Video: How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Aspiración Micropipeta de células adheridas a sustratos para estimar la rigidez Celular

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Investigación

Bioingeniería

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Medición de las propiedades mecánicas de las células vivas mediante microscopía de fuerza atómica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Medida longitudinal de la matriz extracelular Rigidez en modelos de tumores en 3D utilizando partículas de seguimiento Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

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

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

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 ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

El estudio de los efectos de la matriz de rigidez en la función celular usando como base la acrilamida-hidrogeles

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Biología

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Micro-Mecánica Caracterización del tejido pulmonar con microscopía de fuerza atómica

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Un ensayo de base poliacrilamida formato multi-bien para estudiar el efecto de la rigidez de la matriz extracelular en la infección bacteriana de las células adherentes

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Inmunología e Infección

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Microscopía de Fuerza Atómica y Confocal de Escaneo Láser Casi Simultánea (Conpokal) en Células Vivas

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young&#39;s modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young&#39;s modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young&#39;s moduli, and generation of Young&#39;s modulus maps. Finally, it shows how to determine and isolate Young&#39;s modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This protocol visualizes how to prepare cryosections of murine lung tissue, perform atomic force microscopy force map experiments, and analyze the data to determine Young's modulus of the native murine pulmonary basement membrane.

Extracción del módulo de Young de las membranas basales pulmonares murinos nativas de los mapas de fuerza derivados de la microscopía de fuerza atómica

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

La medición de la rigidez de<em&gt; Ex Vivo</em&gt; Ratón aortas Usando microscopía de fuerza atómica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Como estudiar membrana basal para la rigidez como un disparador de Biofísica en el cáncer de próstata y otras patologías relacionadas con la edad o enfermedades metabólicas

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