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

Dette arbejde blev støttet af forskningsstipendier (NRF-2018-R1D1A1B07050175, HURF-2017-66) fra National Research Foundation (NRF) i Korea og Hallym University Research Fund.

Alle forskningsprotokoller med ICR-mus blev godkendt af Institutional Animal Care and Use Committee (IACUC) ved Yonsei University ved Wonju College of Medicine. Eksperimenter blev udført i henhold til kodekset for etik i World Medical Association. I denne protokol blev gravide ICR-mus holdt i en 12/12 h lys / mørk cyklus med fri adgang til mad og vand.
1. Cochleae dissektion
<ol><li>Steriliser laminar flow vævskulturhætten ved at tænde for ultraviolet lys i ~ 30 min, og sprøjt alle ove...

<ol>
	<li>Brown, C. S., Emmett, S. D., Robler, S. K., Tucci, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+hearing+loss+prevention.">Global hearing loss prevention.</a> Otolaryngologic Clinics of North America. 51 (3), 575-592 (2018).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25 (11), 2739-2749 (2007).</li><li>Fu, X., 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+migration+and+tissue+repair.">Mesenchymal stem cell migration and tissue repair.</a> Cells. 8 (8), (2019).</li><li>Uder, C., Br&#252;ckner, S., Winkler, S., Tautenhahn, H. M., Christ, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+MSC+from+selected+species:+Features+and+applications.">Mammalian MSC from selected species: Features and applications.</a> Cytometry A. 93 (1), 32-49 (2018).</li><li>Rojewski, M. T., 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=Translation+of+a+standardized+manufacturing+protocol+for+mesenchymal+stromal+cells:+A+systematic+comparison+of+validation+and+manufacturing+data.">Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data.</a> Cytotherapy. 21 (4), 468-482 (2019).</li><li>Ullah, M., Liu, D. D., Thakor, 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=Mesenchymal+stromal+cell+homing:+Mechanisms+and+strategies+for+improvement.">Mesenchymal stromal cell homing: Mechanisms and strategies for improvement.</a> iScience. 15, 421-438 (2019).</li><li>Ahn, Y. 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. 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. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+neonatal+murine+inner+ear+explants.">Organotypic culture of neonatal murine inner ear explants.</a> Frontiers in Cellular Neuroscience. 13, 170 (2019).</li><li>Oshima, 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=Mechanosensitive+hair+cell-like+cells+from+embryonic+and+induced+pluripotent+stem+cells.">Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells.</a> Cell. 141 (4), 704-716 (2010).</li></ol>

Transplantation af MSC'er til beskadigede steder for at fremme regenerering af beskadigede celler er blevet grundigt undersøgt, og den terapeutiske effekt er tydelig. Transplantation og efterfølgende differentiering af MSC'er er blevet rapporteret for at genoprette hørelsen hos rotter med høretab fremkaldt af 3-nitropropionsyre13. Selv om Lee et al. anvendt MSCs til mennesker trans-venøst, de ikke opnå nogen væsentlig forbedring i retsmødet14. Indtil for nylig blev ...

Høretab kan forekomme medfødt eller kan forårsages gradvist af flere faktorer, herunder aldring, medicin og støj. Høretab er ofte vanskeligt at behandle, fordi det er meget udfordrende at genoprette nedsat funktion, når de hårceller, der er ansvarlige for hørelsen, er beskadiget1. Ifølge Verdenssundhedsorganisationen skønnes 461 millioner mennesker verden over at have høretab, som udgør 6,1% af verdens befolkning. Af dem med høretab er 93 % voksne, og 7 % er børn.
En række tilgange er blevet forsøgt at behandle høretab; navnlig har en regenereringsmetode, hvor MSC'er er blevet brugt, vist sig at være en lovende behandling. Når væv er beskadiget, frigives MSC'er naturligt i kredsløbssystemet og migrerer til skadesstedet, hvor de udskiller forskellige molekyler for at danne et mikromiljø, der fremmer regenerering2. Derfor er det vigtigt at udvikle en metode til behandling af beskadigede væv gennem migration af eksternt implanterede MSC'er til at målrette organer og deres efterfølgende sekretion af molekyler, der forårsager potent immunregulering, angiogenese og antiapoptose for at forbedre genoprettelsen af beskadiget cellefunktion3,4,5.
Den homing proces, hvor MSCs migrere til beskadigede væv kan være den vigtigste hindring for at overvinde. MSC'er har en systemisk sporingsmekanisme med sekventielle trin til tøjring/rulning, aktivering, anholdelse, transmigration/diapedesis og migrering6,7,8. I øjeblikket er der bestræbelser i gang for at finde måder at forbedre disse trin på. Forskellige strategier, herunder genmodifikation, celleoverfladeteknik, in vitro priming og magnetisk vejledning, er blevet testet6,7. Desuden er der gjort adskillige forsøg på at fremme beskyttelse og regenerering af auditive hårceller ved at homing MSCs til det sted, hvor beskadiget cochlea. Sporing af MSC'er in vivo er imidlertid tidskrævende og arbejdskrævende og kræver højt specialiserede færdigheder9.
For at løse dette problem blev der udviklet en metode til at observere homing af MSC'er i cochlea gennem time-lapse konfokal mikroskopi, der fotograferer migration af celler over flere timer (Figur 1). Det blev udviklet i begyndelsen af det20. århundrede og er for nylig blevet et kraftfuldt værktøj til at studere migration af specifikke celler.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> Figur 1: Grafisk abstrakt. (A) Efter at Cortis dissekerede organ er klæbet på en plastovertræksafløb ved hjælp af tang, placeres coverlipet på en 35 mm glasbundet konfokal mikroskopisk skål, og (B) glascylinderen er placeret. (C) Efter påfyldning af indersiden af glascylinderen med medium tilsættes (D) GFP-mærkede MSC'er med medium forsigtigt uden for cylinderen. (E) Efter inkubation natten over fjernes (F) glascylinderen, og billeder tages med et konfokalt mikroskop. Forkortelser: GFP = grønt fluorescerende protein; MSC = mesenchymale stamceller. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">Klik her for at se en større version af dette tal.</a>

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

For at studere virkningerne af mesenchymale stamceller (MSC) på cellegendannelse og -behandling sporer denne metode MSC-migration og morfologiske ændringer efter co-kultur med cochlear epitel. Corti-organet blev immobiliseret på en plastikovertræk ved at trykke på en del af Reissners membran, der blev genereret under dissektionen. MSC'er, der var begrænset af en glascylinder, migrerede mod cochlear epitel, da cylinderen blev fjernet. Deres dominerende lokalisering blev observeret i modiolus af Cortis organ, justeret i en retning svarende til nervefibrene. Nogle MSC'er var imidlertid lokaliseret i limbusområdet og viste en vandret langstrakt form. Derudover blev migrationen til hårcelleområdet øget, og MSC'ernes morfologi ændrede sig til forskellige former efter kanamycinbehandling. Afslutningsvis tyder resultaterne af denne undersøgelse på, at cokulturen af MSC'er med cochlear epitel vil være nyttig for udviklingen af terapeutiske behandlinger via celletransplantation og for undersøgelser af cellegendannelse, der kan undersøge forskellige tilstande og faktorer.

In vitro-migration af MSC'er i tredimensionel tilstand er blevet vurderet af et Transwell-system eller af den traditionelle sårhelingsmetode for at observere migration i todimensionel (2D) tilstand11. Cortis organ er en kompleks struktur bestående af forskellige celler som Boettcher-celler, Claudius-celler, Deiters-celler, søjleceller, Hensens celler, ydre hårceller, indre hårceller, nervefibre, basilarmembran og retikulær lamina12. N?...

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

I denne undersøgelse præsenterer vi en realtidsbilleddannelsesmetode ved hjælp af konfokal mikroskopi for at observere celler, der bevæger sig mod beskadiget væv ved ex vivo-inkubation med cochlear epitel, der indeholder Cortis organ.

In vitro Time-lapse Live-Cell Imaging at udforske Cell Migration mod Orgel 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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In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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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

Gennem Looking Glass: Time-lapse mikroskopi og Longitudinal Tracking af enkelte celler til at studere antikræftmidler

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

Udvidet Time-lapse Intravital Imaging af Real-time flercellede Dynamics i Tumor mikromiljø

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