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

Detta arbete stöddes av forskningsanslag (NRF-2018-R1D1A1B07050175, HURF-2017-66) från National Research Foundation (NRF) i Korea och Hallym University Research Fund.

Alla forskningsprotokoll som omfattade ICR-möss godkändes av Institutional Animal Care and Use Committee (IACUC) vid Yonsei University vid Wonju College of Medicine. Experiment utfördes enligt Världsmedicinska föreningens etiska kodex. I detta protokoll hölls gravida ICR möss i en 12/12 h ljus / mörk cykel med fri tillgång till mat och vatten.
1. Cochleae dissekering
<ol><li>Sterilisera den laminära flödesvävnadskulturhuven genom att slå på ultraviolett ljus i ~ 30 min och spra...

<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 av MSC till skadade platser för att främja regenerering av skadade celler har studerats utförligt, och den terapeutiska effekten är uppenbar. Transplantation och efterföljande differentiering av MSC har rapporterats återställa hörseln hos råttor med hörselnedsättning inducerad av 3-nitropropionsyra13. Även om Lee et al. applicerade MSCs på människor trans-venously, uppnådde de inte någon betydande förbättring i hörsel14. Fram till nyligen ...

Hörselnedsättning kan uppstå medfödda eller kan orsakas gradvis av flera faktorer, inklusive åldrande, droger och buller. Hörselnedsättning är ofta svår att behandla eftersom det är mycket utmanande att återställa nedsatt funktion när hårcellerna som ansvarar för hörseln är skadade1. Enligt Världshälsoorganisationen beräknas 461 miljoner människor världen över ha hörselnedsättning, vilket utgör 6,1 procent av världens befolkning. Av dem med hörselnedsättning är 93 procent vuxna och 7 procent är barn.
Ett antal tillvägagångssätt har försökt behandla hörselnedsättning. I synnerhet har en regenereringsstrategi med hjälp av msc-grupper framkommit som en lovande behandling. När vävnaden skadas frigörs msc naturligt i cirkulationssystemet och migrerar till skadestället där de utsöndrar olika molekyler för att bilda en mikromiljö som främjar regenerering2. Därför är det viktigt att utveckla en metod för att behandla skadade vävnader genom migrering av externt implanterade MSC till målorgan och deras efterföljande utsöndring av molekyler som orsakar potent immunreglering, angiogenes och antiapoptos för att förbättra återställandet av skadad cellfunktion3,4,5.
Den hamstringsprocess där msc migrerar till skadade vävnader kan vara det viktigaste hindret att övervinna. MsCs har en systemisk homing mekanism med sekventiella steg av tjudring / rullande, aktivering, gripande, transmigration / diapedesis ochmigrering 6,7,8. För närvarande pågår ansträngningar för att identifiera sätt att förbättra dessa steg. Olika strategier, inklusive genetisk modifiering, cellytasteknik, in vitro-priming och magnetisk vägledning, har testats6,7. Dessutom har flera försök gjorts att främja skydd och regenerering av hörselhårceller genom att föra msc till platsen för skadad cochlea. Att spåra MSCs in vivo är dock tidskrävande och arbetsintensivt och kräver högspecialiserade färdigheter9.
För att lösa detta problem utvecklades en metod för att observera hamstring av MSC i cochlea genom tidsfördröjning konfokal mikroskopi som fotograferar migreringen av celler under flera timmar (Figur 1). Det utvecklades i början av 1900-talet och har nyligen blivit ett kraftfullt verktyg för att studera migration av specifika celler.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> Bild 1: Grafiskt abstrakt. (A) Efter att Cortis dissekerade organ har fästs på ett plastlock med tång placeras täcket på en 35 mm glasbottnad konfokal mikroskopisk skål, och (B) glascylindern är placerad. c)Efter att ha fyllt glascylinderns insida med medium tillsätts (D) GFP-märkta MSC med medium försiktigt utanför cylindern. ( E) Efter inkubation över natten,(F) tas glascylindern bort och bilder tas med ett konfokalt mikroskop. Förkortningar: GFP = grönt fluorescerande protein; MSC = mesenchymala stamceller. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">Klicka här om du vill visa en större version av den här figuren.</a>

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			<td>P2100-104</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>
			<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>
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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>
			<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>
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			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
			<td></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

För att studera effekterna av mesenkymala stamceller (MSC) på cellregenerering och behandling spårar denna metod MSC-migration och morfologiska förändringar efter samkultur med cochlear epitel. Cortis organ immobiliserades på en plastlock genom att trycka på en del av Reissners membran som genererades under dissekeringen. MsCs begränsas av en glascylinder migrerade mot cochlear epitel när cylindern togs bort. Deras dominerande lokalisering observerades i modiolus av organet Corti, i linje i en riktning som liknar nervfibrerna. Vissa MSCs var dock lokaliserade i limbus området och visade en horisontellt långsträckt form. Dessutom ökades migrationen till hårcellsområdet, och morfologin hos MSCs ändrades till olika former efter kanamycinbehandling. Sammanfattningsvis visar resultaten av denna studie att kokultur av MSC med cochlear epitel kommer att vara användbart för utveckling av terapier via celltransplantation och för studier av cellregenerering som kan undersöka olika tillstånd och faktorer.

In vitro-migrering av msc i tredimensionellt läge har bedömts av ett Transwell-system eller med den traditionella sårläkningsmetoden för att observera migration i tvådimensionellt (2D) läge11. Cortis organ är en komplex struktur som består av olika celler som Boettcher-celler, Claudius-celler, Deitersceller, pelarceller, Hensens celler, yttre hårceller, inre hårceller, nervfibrer, basilarmembran och retikulär lamina12. När msc-?...

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 denna studie presenterar vi en realtidsavbildningsmetod med konfokal mikroskopi för att observera celler som rör sig mot skadad vävnad genom ex vivo inkubation med cochlear epitel som innehåller Cortis organ.

In vitro Time-lapse Live-Cell Imaging för att utforska cellmigration mot Cortis organ

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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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

Utökad Time-lapse Intravital avbildning av realtid flercelliga Dynamics i tumörens mikro

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