This work was supported by research grants (NRF-2018-R1D1A1B07050175, HURF-2017-66) from the National Research Foundation (NRF) of Korea and Hallym University Research Fund.

All research protocols involving ICR mice were approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University at Wonju College of Medicine. Experiments were performed according to the Code of Ethics of the World Medical Association. In this protocol, pregnant ICR mice were kept in a 12/12 h light/dark cycle with free access to food and water.
1. Cochleae dissection
<ol>
	<li>Sterilize the laminar flow tissue culture hood by turning on the ultraviolet light for ~30 m...

<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 of MSCs into damaged sites to promote the regeneration of damaged cells has been extensively studied, and the therapeutic effect is evident.The transplantation and subsequent differentiation of MSCs have been reported to restore hearing in rats with hearing loss induced by 3-nitropropionic acid13. Although Lee et al.&#160;applied MSCs to human beings trans-venously, they did not achieve any significant improvement in hearing14. Until recently, nearly 12 expe...

Hearing loss can occur congenitally or can be caused progressively by several factors, including aging, drugs, and noise. Hearing loss is often difficult to treat because it is very challenging to restore impaired function once the hair cells responsible for hearing are damaged1. According to the World Health Organization, 461 million people worldwide are estimated to have hearing loss, which accounts for 6.1% of the world&#39;s population. Of those with hearing loss, 93% are adults, and 7% are children.
A number of approaches have been attempted to treat hearing loss; notably, a regeneration approach using MSCs has emerged as a promising treatment. When tissue is damaged, MSCs are naturally released into the circulatory system and migrate to the injury site where they secrete various molecules to form a microenvironment that promotes regeneration2. Hence, it is important to develop a method to treat damaged tissues through the migration of externally implanted MSCs to target organs and their subsequent secretion of molecules that cause potent immune regulation, angiogenesis, and anti-apoptosis to enhance the restoration of damaged cell function3,4,5.
The homing process in which MSCs migrate to damaged tissues may be the most important obstacle to overcome. MSCs have a systemic homing mechanism with sequential steps of tethering/rolling, activation, arrest, transmigration/diapedesis, and migration6,7,8. Currently, efforts are underway to identify ways to improve these steps. Various strategies, including genetic modification, cell surface engineering, in vitro priming, and magnetic guidance, have been tested6,7. In addition, several attempts have been made to promote the protection and regeneration of auditory hair cells by homing MSCs to the site of damaged cochlea. However, tracking MSCs in vivo is time-consuming and labor-intensive and requires highly specialized skills9.
To solve this problem, a method was developed to observe the homing of MSCs in the cochlea through time-lapse confocal microscopy that photographs the migration of cells over several hours (Figure 1). It was developed in the early 20th century and has recently become a powerful tool for studying migration of specific cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;" /> 
Figure 1: Graphical abstract. (A)&#160;After the dissected organ of Corti is adhered on a plastic coverslip using forceps, the coverslip is placed on a 35 mm glass-bottomed confocal microscopic dish, and (B) the glass cylinder is positioned. (C) After filling the inside of the glass cylinder with medium, (D) GFP-labeled MSCs with medium are added carefully outside the cylinder. (E) After incubation overnight, (F) the glass cylinder is removed, and images are taken with a confocal microscope. Abbreviations: GFP = green fluorescent protein; MSCs = mesenchymal stem cells.&#160;<a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		<tr>
			<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>
		<tr>
			<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>
		<tr>
			<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>
</table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In vitro migration of MSCs in three-dimensional mode has been assessed by a Transwell system or by the traditional wound healing method to observe migration in two-dimensional (2D) mode11. The organ of Corti is a complex structure composed of various cells such as Boettcher cells, Claudius cells, Deiters cells, pillar cells, Hensen&#39;s cells, outer hair cells, inner hair cells, nerve fibers, basilar membrane, and reticular lamina12. When M...

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

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of 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.

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

Abstract