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 trabalho foi apoiado por bolsas de pesquisa (NRF-2018-R1D1A1B07050175, HURF-2017-66) da National Research Foundation (NRF) da Coreia e do Hallym University Research Fund.

Todos os protocolos de pesquisa envolvendo camundongos ICR foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) da Universidade yonsei da Faculdade de Medicina wonju. Os experimentos foram realizados de acordo com o Código de Ética da Associação Médica Mundial. Neste protocolo, os camundongos gestantes de ICR foram mantidos em um ciclo claro/escuro de 12/12 horas com livre acesso a alimentos e água.
1. Dissecção de cochleae
<ol><li>Esterilize a capa de cult...

<ol>
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D., Dilwali, S., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+murine+cochlear+explant+technique+as+an+in+vitro+screening+tool+in+hearing+research.">Neonatal murine cochlear explant technique as an in vitro screening tool in hearing research.</a> Journal of Visualized Experiments. (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. 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O transplante de MSCs em locais danificados para promover a regeneração de células danificadas tem sido extensivamente estudado, e o efeito terapêutico é evidente. O transplante e a subsequente diferenciação dos MSCs foram relatados para restaurar a audição em ratos com perda auditiva induzida por ácido 3-nitropropídico13. Embora Lee et al. aplicassem MSCs aos seres humanos transvenosamente, eles não alcançaram qualquer melhora significativa na audição14. At?...

A perda auditiva pode ocorrer congênitamente ou pode ser causada progressivamente por vários fatores, incluindo envelhecimento, drogas e ruído. A perda auditiva é muitas vezes difícil de tratar porque é muito desafiador restaurar a função prejudicada uma vez que as células ciliadas responsáveis pela audição são danificadas1. De acordo com a Organização Mundial da Saúde, estima-se que 461 milhões de pessoas em todo o mundo tenham perda auditiva, o que representa 6,1% da população mundial. Dos que têm perda auditiva, 93% são adultos e 7% são crianças.
Várias abordagens foram tentadas para tratar a perda auditiva; notavelmente, uma abordagem de regeneração usando MSCs emergiu como um tratamento promissor. Quando o tecido é danificado, os MSCs são naturalmente liberados para o sistema circulatório e migram para o local da lesão onde secretam várias moléculas para formar um microambiente que promove a regeneração2. Por isso, é importante desenvolver um método para tratar tecidos danificados através da migração de MSCs implantados externamente para órgãos alvo e sua subsequente secreção de moléculas que causam regulação imunológica potente, angiogênese e anti-apoptose para melhorar a restauração da função celular danificada3,4,5.
O processo de homing no qual os MSCs migram para tecidos danificados pode ser o obstáculo mais importante a ser superado. Os MSCs possuem um mecanismo de vagem sistêmica com etapas sequenciais de amarração/rolagem, ativação, prisão, transmigração/diápedese e migração6,7,8. Atualmente, estão em andamento esforços para identificar formas de melhorar essas etapas. Várias estratégias, incluindo modificação genética, engenharia de superfície celular, escoramento in vitro e orientação magnética, foram testadas6,7. Além disso, várias tentativas foram feitas para promover a proteção e regeneração de células ciliadas auditivas, lançando MSCs para o local da cóclea danificada. No entanto, o rastreamento de MSCs in vivo é demorado e trabalhoso e requer habilidades altamente especializadas9.
Para resolver esse problema, foi desenvolvido um método para observar o homing de MSCs na cóclea através de microscopia confocal de lapso de tempo que fotografa a migração de células ao longo de várias horas(Figura 1). Foi desenvolvido no início do séculoXX e recentemente tornou-se uma poderosa ferramenta para estudar a migração de células específicas.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> Figura 1: Resumo gráfico. (A) Depois que o órgão dissecado de Corti é aderido a um deslizamento de tampas de plástico usando fórceps, o deslizamento de tampa é colocado em um prato microscópico de 35 mm com fundo de vidro, e(B)o cilindro de vidro é posicionado. (C) Depois de encher o interior do cilindro de vidro com MSCs médios,(D)com etiqueta GFP com meio são adicionados cuidadosamente fora do cilindro. (E) Após a incubação durante a noite,(F) o cilindro de vidro é removido, e as imagens são tiradas com um microscópio confocal. Abreviaturas: GFP = proteína fluorescente verde; MSCs = células-tronco mesenquimais. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

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			<td>10X PBS Buffer</td>
			<td>GenDEPOT</td>
			<td>P2100-104</td>
			<td></td>
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		<tr>
			<td>4% Formalin</td>
			<td>T&#38;I</td>
			<td>BPP-9004</td>
			<td></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>
			<td></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>
			<td></td>
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			<td>Fluorsheild with DAPI</td>
			<td>sigma&#160;</td>
			<td>F6057</td>
			<td></td>
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			<td>Forcep</td>
			<td>Dumont</td>
			<td>0508-L5-P0</td>
			<td></td>
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		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher scientific</td>
			<td>14065056</td>
			<td></td>
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			<td>HEPES</td>
			<td>Thermo Fisher scientific</td>
			<td>15630080</td>
			<td></td>
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			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
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			<td>Phalloidin-iFluor 647 Reagent</td>
			<td>abcam</td>
			<td>ab176759</td>
			<td></td>
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		<tr>
			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td>
			<td>cyagen</td>
			<td>MUBMX-01101</td>
			<td></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 estudar os efeitos das células-tronco mesenquimais (MSCs) na regeneração e tratamento celular, este método acompanha a migração do MSC e as alterações morfológicas após a cocultura com epitélio coclear. O órgão de Corti foi imobilizado em uma mancha de plástico pressionando uma porção da membrana do Reissner gerada durante a dissecação. MSCs confinados por um cilindro de vidro migraram em direção ao epitélio coclear quando o cilindro foi removido. Sua localização predominante foi observada no modiol do órgão de Corti, alinhado em uma direção semelhante à das fibras nervosas. No entanto, alguns MSCs foram localizados na área do limbus e apresentaram uma forma horizontalmente alongada. Além disso, a migração para a área da célula ciliada foi aumentada, e a morfologia dos MSCs mudou para várias formas após o tratamento da kanamicina. Concluindo, os resultados deste estudo indicam que a cocultura de MSCs com epitélio coclear será útil para o desenvolvimento da terapêutica via transplante celular e para estudos de regeneração celular que possam examinar várias condições e fatores.

A migração in vitro de MSCs no modo tridimensional foi avaliada por um sistema Transwell ou pelo método tradicional de cicatrização de feridas para observar a migração no modo bidimensional (2D)11. O órgão de Corti é uma estrutura complexa composta por várias células como células Boettcher, células de Cláudio, células de Deiters, células pilares, células de Hensen, células ciliar externas, células ciliar internas, fibras nervosas, membrana manje...

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

Neste estudo, apresentamos um método de imagem em tempo real utilizando microscopia confocal para observar células que se movem em direção ao tecido danificado pela incubação ex vivo com o epitélio coclear contendo o órgão de Corti.

Imagens de células vivas in vitro para explorar a migração celular em direção ao órgão 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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Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Through the Looking Glass: Microscopia Time-lapse e Longitudinal de rastreamento de células individuais para estudar Therapeutics Anti-câncer

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

Estendida Time-lapse Imagem intravital do Real-time multicelular Dynamics no microambiente tumoral

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