Name: in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti
Uploaded: 2020-12-04T14:45:00
Description: Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

Este trabajo fue apoyado por becas de investigación (NRF-2018-R1D1A1B07050175, HURF-2017-66) de la Fundación Nacional de Investigación (NRF) de Corea y el Fondo de Investigación de la Universidad Hallym.

Todos los protocolos de investigación que involucran ratones ICR fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de Yonsei en wonju Facultad de Medicina. Los experimentos se realizaron de acuerdo con el Código de Ética de la Asociación Médica Mundial. En este protocolo, los ratones embarazadas de ICR fueron mantenidos en un ciclo claro/oscuro de 12/12 h con el acceso libre al alimento y al agua.
1. Disección de cocleas
<ol><li>Esteri...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+enhance+efficacy+of+SPION-labeled+stem+cell+homing+by+magnetic+attraction:+a+systemic+review+with+meta-analysis.">Strategies to enhance efficacy of SPION-labeled stem cell homing by magnetic attraction: a systemic review with meta-analysis.</a> International Journal of Nanomedicine. 14, 4849-4866 (2019).</li><li>Alon, R., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+on+the+run:+shear-regulated+integrin+activation+in+leukocyte+rolling+and+arrest+on+endothelial+cells.">Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells.</a> Current Opinion in Cell Biology. 20 (5), 525-532 (2008).</li><li>Sykova, E., Jendelova, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tracking+of+stem+cells+in+brain+and+spinal+cord+injury.">In vivo tracking of stem cells in brain and spinal cord injury.</a> Progress in Brain Research. 161, 367-383 (2007).</li><li>Landegger, L. 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(124), e55704 (2017).</li><li>Pijuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration,+invasion,+and+adhesion+assays:+From+cell+imaging+to+data+analysis.">In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis.</a> Frontiers in Cell and Developmental Biology. 7, 107 (2019).</li><li>Rask-Andersen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cochlea:+anatomical+characteristics+and+their+relevance+for+cochlear+implantation.">Human cochlea: anatomical characteristics and their relevance for cochlear implantation.</a> The Anatomical Record. 295 (11), 1791-1811 (2012).</li><li>Kamiya, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+transplantation+accelerates+hearing+recovery+through+the+repair+of+injured+cochlear+fibrocytes.">Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.</a> The American Journal of Pathology. 171 (1), 214-226 (2007).</li><li>Lee, H. S., Kim, W. J., Gong, J. S., Park, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+safety+and+efficacy+of+autologous+bone+marrow-derived+mesenchymal+stem+cell+transplantation+in+sensorineural+hearing+loss+patients.">Clinical safety and efficacy of autologous bone marrow-derived mesenchymal stem cell transplantation in sensorineural hearing loss patients.</a> Journal of Audiology and Otology. 22 (2), 105-109 (2018).</li><li>Vanden Berg-Foels, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+tissue+regeneration:+chemoattractants+for+endogenous+stem+cell+recruitment.">In situ tissue regeneration: chemoattractants for endogenous stem cell recruitment.</a> Tissue Engineering Part B: Reviews. 20 (1), 28-39 (2014).</li><li>Parker, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> Journal of Visualized Experiments. (36), e1685 (2010).</li><li>Ogier, J. M., Burt, R. A., Drury, H. R., Lim, R., Nayagam, B. 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El trasplante de MSCs en sitios dañados para promover la regeneración de células dañadas se ha estudiado extensivamente, y el efecto terapéutico es evidente. El trasplante y la diferenciación subsecuente de MSCs se han divulgado para restaurar la audiencia en ratas con la pérdida de oído inducida por el ácido 3-nitropropionic13. Aunque Lee et al. aplicaron mscs a seres humanos transvenosamente, no lograron ninguna mejora significativa en la audición14. Hasta hace ...

La pérdida de audición puede ocurrir congénitamente o puede ser causada progresivamente por varios factores, incluyendo el envejecimiento, las drogas y el ruido. La pérdida de audición es a menudo difícil de tratar porque es muy difícil restaurar la función deteriorada una vez que las células ciliadas responsables de la audición están dañadas1. Según la Organización Mundial de la Salud, se estima que 461 millones de personas en todo el mundo tienen pérdida auditiva, lo que representa el 6,1% de la población mundial. De las personas con pérdida auditiva, el 93% son adultos y el 7% son niños.
Se han intentado varios enfoques para tratar la pérdida de audición; notablemente, un acercamiento de la regeneración usando MSCs ha emergido como tratamiento prometedor. Cuando el tejido está dañado, los MSCs se liberan naturalmente en el sistema circulatorio y migran al sitio de la lesión donde secretan varias moléculas para formar un microambiente que promueve la regeneración2. Por lo tanto, es importante desarrollar un método para tratar los tejidos dañados a través de la migración de mscs implantados externamente a los órganos diana y su posterior secreción de moléculas que causan una potente regulación inmune, angiogénesis y anti-apoptosis para mejorar la restauración de la función celular dañada3,4,5.
El proceso de búsqueda en el que los MSCs migran a los tejidos dañados puede ser el obstáculo más importante a superar. Las MSCs tienen un mecanismo de localización sistémico con pasos secuenciales de atado/balanceo, activación, detención, transmigración/diapedesis y migración6,7,8. Actualmente, se están realizando esfuerzos para identificar formas de mejorar estas medidas. Se han ensayado diversas estrategias, entre ellas la modificación genética, la ingeniería de la superficie celular, el cebado in vitro y la orientación magnética6,7. Además, varias tentativas se han hecho de promover la protección y la regeneración de células de pelo auditivas homing MSCs al sitio de la cóclea dañada. Sin embargo, el seguimiento de las CSS in vivo requiere mucho tiempo y mano de obra y requiere habilidades altamente especializadas9.
Para resolver este problema, se desarrolló un método para observar el homing de MSCs en la cóclea a través de microscopía confocal time-lapse que fotografía la migración de las células durante varias horas (Figura 1). Fue desarrollado a principios delsiglo 20 y recientemente se ha convertido en una poderosa herramienta para estudiar la migración de células específicas.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> Figura 1:Resumen gráfico. (A) Después de que el órgano diseccionado de Corti se adhiere en una cubierta de plástico usando fórceps, la tapa se coloca en un plato microscópico confocal con fondo de vidrio de 35 mm, y(B) se coloca el cilindro de vidrio. (C) Después de llenar el interior del cilindro de vidrio con medio, (D) los MSC etiquetados con GFP con medio se agregan cuidadosamente fuera del cilindro. (E) Después de la incubación durante la noche, (F) se retira el cilindro de vidrio, y las imágenes se toman con un microscopio confocal. Abreviaturas: GFP = proteína fluorescente verde; MSCs = células madre mesenquimales. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">Haga clic aquí para ver una versión más amplia de esta figura.</a>

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			<td>GenDEPOT</td>
			<td>P2100-104</td>
			<td></td>
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			<td>4% Formalin</td>
			<td>T&#38;I</td>
			<td>BPP-9004</td>
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			<td>Ampicillin</td>
			<td>sigma&#160;</td>
			<td>A5354-10ml</td>
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			<td>BSA</td>
			<td>sigma&#160;</td>
			<td>A4503-100G</td>
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			<td>confocal dish</td>
			<td>SPL</td>
			<td>200350</td>
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			<td>confocal microscope&#160;</td>
			<td>ZEISS</td>
			<td>LSM800</td>
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			<td>coverslip</td>
			<td>SPL</td>
			<td>20009</td>
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			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
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			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher scientific</td>
			<td>16140071</td>
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			<td>Fluorsheild with DAPI</td>
			<td>sigma&#160;</td>
			<td>F6057</td>
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			<td>Forcep</td>
			<td>Dumont</td>
			<td>0508-L5-P0</td>
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			<td>HBSS</td>
			<td>Thermo Fisher scientific</td>
			<td>14065056</td>
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			<td>HEPES</td>
			<td>Thermo Fisher scientific</td>
			<td>15630080</td>
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			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
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			<td>Phalloidin-iFluor 647 Reagent</td>
			<td>abcam</td>
			<td>ab176759</td>
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			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
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			<td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td>
			<td>cyagen</td>
			<td>MUBMX-01101</td>
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			<td>Triton X-100</td>
			<td>sigma&#160;</td>
			<td>T8787</td>
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in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

Para estudiar los efectos de las células madre mesenquimales (MSC) en la regeneración celular y el tratamiento, este método rastrea la migración msc y los cambios morfológicos después de co-cultivo con epitelio coclear. El órgano de Corti fue inmovilizado en una cubierta plástica presionando una porción de la membrana del Reissner generada durante la disección. MsCs confinado por un cilindro de cristal migró hacia el epitelio coclear cuando el cilindro fue quitado. Su localización predominante se observó en el modiolus del órgano de Corti, alineado en una dirección similar a la de las fibras nerviosas. Sin embargo, algunos MSCs fueron localizados en el área del limbus y demostraron una forma horizontalmente alargada. Además, la migración en el área de la célula de pelo fue aumentada, y la morfología del MSCs cambió a las varias formas después del tratamiento del kanamycin. En conclusión, los resultados de este estudio indican que el cocultivo de MSCs con epitelio coclear será útil para el desarrollo de la terapéutica vía el trasplante de la célula y para los estudios de la regeneración de la célula que pueden examinar las varias condiciones y factores.

La migración in vitro de mscs en modo tridimensional ha sido evaluada por un sistema Transwell o por el método tradicional de cicatrización de heridas para observar la migración en modo bidimensional (2D)11. El órgano de Corti es una estructura compleja compuesta por varias células como las células de Boettcher, las células de Claudio, las células de Deiters, las células del pilar, las células de Hensen, las células ciliadas externas, las células cilia...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

En este estudio, presentamos un método de la proyección de imagen en tiempo real usando microscopia confocal para observar las células que se mueven hacia el tejido dañado por la incubación ex vivo con el epitelio coclear que contiene el órgano de Corti.

In vitro Time-lapse live-cell imaging para explorar la migración celular hacia el órgano de Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Existing in vitro method to investigate migration of MSCs include Transwell or wound healing protocols. The method described here can be used to track migration of MSCs to explant in real time. In terms of culture of explant, the advantage of this method compared to others is that it achieves a reduction in time and cost, as well as a simplification of experimental steps.This technique can be used to observe stem cell homing to target in real time leading to development of models with optimized homing efficiency. Removal of tectorial membrane is difficult for unexperienced researchers. Depending on the development stage, understanding the right time to remove the tectorial membrane is important for the success of this protocol.Demonstrating the procedure will be Dr.Jeong-Eun Park and Dr.Su Hoon Lee, researcher professors from my lab. Begin by sterilizing the laminar flow tissue culture hood by turning on the ultraviolet light for approximately 30 minutes. Spray all surfaces with 70%ethanol prior to use and allow them to dry.Place dissection instruments in 70%ethanol for 10 minutes, then dry them before using. After decapitating a three to four-day-old mouse, soak the ahead in 70%ethanol and place it under a serial microscope in the laminar flow hood. Quickly soak the tissue in tissue dissection solution to remove the ethanol.Cut the center line of the skull with the surgical blade, then expose the skull by pulling down the skin anteriorly and cutting the external auditory canal of the ear. Cut from the anterior to the posterior part of the cranium across the eye line, then open the skull and remove the forebrain, cerebellum, and brainstem with blunt forceps. Using microforceps, separate the cochlea from the temporal bone and transfer it to a Petri dish containing tissue dissection solution.Carefully dissect all of the cochlear otic capsule leaving only the internal cochlear soft tissue. Hold the modiolus of the cochlea with forceps and the cochlear duct with another pair of forceps, then slowly separate the two tissues. Remove the stria vascularis and tectorial membrane by gently peeling them away.Place a sterilized plastic coverslip in new tissue dissection solution, then place the organ of Corti on the coverslip making sure that the basilar membrane faces downward. Immobilize the tissue by pressing the Reissner's membrane and the remaining modiolus tissue onto the coverslip with forceps. Transfer the coverslip with the embedded tissue to the center of a 35 millimeter confocal dish.Place the glass cloning cylinder on the dish with the cochlear explant positioned in the center of the dish and add 100 microliters of explant culture medium into the cylinder. Place 5, 000 bone marrow derived GFP-tagged mesenchymal stem cells or MSCs in two milliliters of culture medium outside the glass cylinder and put the dish in the incubator. After culturing the cells as described in the text manuscript, aspirate all medium inside and outside the cylinder and remove the glass cylinder from the confocal dish.Add two milliliters of fresh culture medium to the dish. Place it in a humidified incubator until ready for analysis. Use a confocal microscopy system with a stage top incubator.Turn on the confocal microscope, fluorescent light and computer. Set the conditions of the stage top incubator to 37 degrees Celsius and 5%carbon dioxide atmosphere. Place the sample dish on the dish fixing vessel, cover it with the dish fixing lid and close the chamber with the top heater lid.Adjust the zoom and focus to localize the organ of Corti and MSCs in the field of view. Open the image processing software. Under the locate option, select a 20X plan-apochromat objective or numerical aperture 0.8 and a 0.5X crop area.Under acquisition, click on smart setup and select EGFP. Open the channel tab under acquisition and set the laser power to 0.2%the pinhole to 44 micrometers, the master gain to 750 volts, and the digital gain to 1.0. Click on ESID under imaging setup and set the ESID gain to four and the digital gain to 7.5.Click on tiles and stake to produce 210 tiles. Open focus strategy and select the focus mode. Under time series, set the duration to 24 hours and the interval to 10 minutes.Under acquisition, set the frame size to 512 by 512 pixels, scan speed to eight, the direction to bidirectional, the averaging to 4X, and the bits per pixel to 16. Click on start experiment to begin the experiment. The two-dimensional path of mesenchymal stem cells or MSCs was tracked in a wound healing method by using a glass cylinder as a barrier between the MSCs and the organ of Corti.When the organ of Corti was cultured, fibroblasts grew very quickly outward from the outer hair cells in the outermost layer. Most GFP-labeled MSCs were pushed out by fast-growing fibroblasts. Nonetheless, some penetrated the layer of fibroblasts and successfully migrated to the organ of Corti.The results from 72 hours of incubation showed that MSCs migrated to the organ of Corti and were mainly localized in the modiolus and the area with the cochlear nerve fibers separated from the modiolus. The morphology of the MSCs was altered to a radial shape similar to that of nerve fibers. The cells were transformed into a linear shape along the limbus line, rather than the hair cell area.The MSCs were able to migrate and grow in the nerve fiber collection area by penetrating the various cell layers and physical barriers. When damage to hair cells was induced by a 16-hour treatment with kanamycin and cells were cultured for a further 72 hours after the removal of kanamycin, MSCs were localized not only in the modiolus, but also in the outer hair cells. Immobilizing the tissue is an important step in this protocol was the organ of Corti is attached.It does not fall off easily. This is a trivial, but helpful tip for successful explant culture. This method may be adapted to study the efficacy of exosomes secreted from MSCs.

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Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

A través del espejo: Microscopía de lapso de tiempo y de seguimiento longitudinal de las células individuales para el Estudio de la terapéutica contra el cáncer

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Prolongado lapso de tiempo intravital de imágenes de tiempo real multicelular Dinámica en el microambiente tumoral

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...