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 min, and spray all surfaces with 70% ethanol prior to use. Allow the surfaces to dry.</li>
	<li>Place dissection instruments in 70% ethanol for 10 min, and dry before using.</li>
	<li>Use an operating blade to decapitate postnatal 3-4 day-old mice (Figure 2A).</li>
	<li>Place the skull under a stereomicroscope in the laminar flow hood, and soak the tissue in 70% ethanol.</li>
	<li>Quickly soak the tissue in tissue dissection solution (1x Hank&#39;s Balanced Salt Solution, 1 mM HEPES) to remove the ethanol.</li>
	<li>Cut the centerline of the skull with a surgical blade (Figure 2B,C).</li>
	<li>Expose the skull by pulling down the skin anteriorly and cutting the external auditory canal of the ear (Figure 2D).</li>
	<li>Cut from the anterior to the posterior part of the cranium across the eye line (Figure 2E).</li>
	<li>Open the skull and remove the forebrain, cerebellum, and brainstem with blunt forceps (Figure 2F,G).</li>
	<li>Using micro forceps, separate the cochlea from the temporal bone (Figure 2H).</li>
	<li>Transfer the cochleae to a Petri dish containing tissue dissection solution.</li>
	<li>Carefully dissect all of the cochlear otic capsule, leaving only the internal cochlear soft tissue (Figure 2I,J).</li>
	<li>Hold the modiolus of the cochleae with forceps and the cochlear duct with another pair of forceps, and slowly separate the two tissues (Figure 2K).</li>
	<li>Remove the stria vascularis and tectorial membrane by gently peeling them away (Figure 2L,M).</li>
	<li>Place a sterilized plastic coverslip in new tissue dissection solution, and then place the organ of Corti on a coverslip of 9 mm diameter, making sure that the basilar membrane faces downward (Figure 2N-P).</li>
	<li>Immobilize the tissue by pressing the Reissner&#39;s membrane and the remaining modiolus tissue onto the coverslip with forceps (Figure 2N-P).</li>
	<li>Transfer the coverslip with the embedded tissue to the center of a confocal dish of 35 mm diameter.</li>
	<li>Place the glass cloning cylinder on the dish, with the cochlear explant positioned in the center of the dish, and add 100 &#181;L of explant culture medium (DMEM/F12, 10% fetal bovine serum (FBS), 1% N2 supplement, ampicillin (10 &#181;g/mL))10 inside the cylinder (Figure 2Q).</li>
	<li>Plate 5 &#215; 103 cells of mouse bone marrow-derived green fluorescent protein (GFP)-tagged MSCs in 2 mL of culture medium (45% DMEM + 45% DMEM/F12, 10% FBS, 1% N2 supplement, 10 &#181;g/mL of ampicillin) outside the glass cylinder (Figure 2R).
	<ol>
		<li>When the MSCs are 80-90% confluent, passage them by detaching them with trypsin-ethylenediamine tetraacetic acid.</li>
	</ol>
	</li>
	<li>Carefully transfer the confocal dish to a humidified incubator and incubate overnight at 37 &#176;C in a 5% CO2 atmosphere.</li>
	<li>Aspirate all medium inside and outside the cylinder, and then remove the glass cylinder from the confocal dish.</li>
	<li>Add 2 mL of fresh culture medium to the confocal dish, and incubate the tissue culture dish in a humidified incubator until ready for analysis.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61947/61947fig2v2.jpg" /> 
Figure 2. Dissection of a mouse cochlea and coculture of the organ of Corti and MSCs. (A) Decapitation of mouse, (B) and (C) midline sagittal dissection of the head, (D) and (E) coronal dissection of the brain, (F) and (G) removal of the brain and temporal bone, (H) the cochlea, (I) removal of the bony cochlear wall, (J) isolation of the cochlea, (K) separation of the cochlear duct from the modiolus, (L) separation of stria vascularis (SV) and spiral ligament (SL) from the organ of Corti, (M) removal of the tectorial membrane, (N-P) fixation of the cochlea on a plastic cover slip, (Q) location of coverslip and glass cylinder in the confocal dish, (R) inoculation of MSCs. White scale bar (A-E) = 1 cm; orange (F, G, P) and yellow scale bar (H,I) = 1 mm; green scale bar (J-O) = 0.5 mm.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61947/61947fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Time-lapse imaging
<ol>
	<li>For the experiments presented here, use a confocal microscopy system with a stage top incubator system.</li>
	<li>Turn on the confocal microscope, fluorescent light, and computer.</li>
	<li>Set the conditions of the stage-top incubator placed on the stage of the confocal microscope to 37 &#176;C and 5% CO2 atmosphere.</li>
	<li>Place the sample dish on the dish fixing vessel, cover with the dish fixing lid, and close the chamber with the top heater lid.</li>
	<li>Adjust the zoom and focus to localize the organ of Corti and MSCs in the field of view.</li>
	<li>Open the image processing software. Under the locate option, select a 20x Plan-Apochromat objective (numerical aperture 0.8) and a 0.5x crop area.</li>
	<li>Under Acquisition, click on smart setup and select EGFP.</li>
	<li>Open the channel tab under Acquisition, and set the laser power to 0.2%, the pinhole to 44 &#181;m, the master gain to 750 V, and the digital gain to 1.0.</li>
	<li>Click on ESID under imaging setup, and set the ESID gain to 4 and the digital gain to 7.5.</li>
	<li>Click on Tiles and stake to produce 210 tiles.</li>
	<li>Open Focus strategy and select the focus mode.</li>
	<li>Under time series, set the duration to 24 h and the interval to 10 min.</li>
	<li>Under Acquisition, set the frame size to 512 x 512 pixels, scan speed to 8, the direction to bidirectional, the averaging to 4x, and the bits per pixel to 16.</li>
	<li>Click on start experiment to begin the experiment.</li>
</ol>
3. Image file modification
<ol>
	<li>Under processing, click on stitching, and set the minimal overlay to 5% and the maximal shift to 10%.</li>
	<li>Click on movie export, set uncompressed, and set the speed to 7.5.</li>
</ol>
4. Immunostaining
<ol>
	<li>Aspirate the medium carefully, and wash the sample twice with phosphate-buffered saline (PBS) for 5 min.</li>
	<li>Fix the sample with 4% formalin in PBS for 15 min, and wash the sample 3 times with PBS for 5 min.</li>
	<li>Permeabilize the sample in 0.1% Triton X-100 in PBS for 10 min, and wash 3 times with PBS for 5 min.</li>
	<li>Add 250 &#181;L of phalloidin-iFluor 647 reagent (1:1000 dilution in PBS), and incubate the sample for 1 h at room temperature on the shaker.</li>
	<li>Wash the sample 3 times with PBS for 5 min.</li>
	<li>Transfer the coverslip onto the glass slide, and add 2 drops of mounting solution.</li>
	<li>Place a coverslip on the slide gently.</li>
	<li>Seal the coverslip with clear nail polish and store at 4 &#176;C in the dark until cells are observed.</li>
	<li>Image the slide using a confocal microscope with an appropriate filter at excitation/emission (Ex/Em)=650/665 nm for phalloidin and at Ex/Em=488/507 nm for EGFP.</li>
</ol>

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The authors have nothing to disclose.

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 experiments were conducted to restore hearing in the rodent model by MSC transplantation. Although the result is somewhat unclear because of heterogeneity, there is some indication that MSCs can promote hearing.
Various growth factors and chemokines related to chemoattraction are involved in the migration of transplanted stem cells to damaged areas, regardless of heterogeneity or homogeneity15. Chemokines are key regulators of cell migration for tissue homeostasis, immune responses, and wound healing. Chemokine-induced chemoattraction in vivo might not work in the same way as it does in vitro due to the differences between in vivo and in vitro experimental conditions. Although in vitro experimental conditions have their own limitations, it is very difficult to conduct in vivo studies to promote efficient migration, recruitment, and regeneration of cells16.
Although the tracking of MSCs transplanted for hearing restoration is important in regenerative medicine research, in vivo experiments, especially those involving hearing, are labor-intensive in most cases and are therefore extremely difficult to perform with large sample sizes. Hence, the homogeneity of the results is low, and the bias is severe. Therefore, it would be efficient to first study such migration-based events under in vitro conditions. Most in vitro studies on cell migration have been performed by using Transwell or wound healing methods.
A coincubation method of explanting cochleae with MSCs was established to track the migrating stem cells, which recognize injury sites and move toward them. Most studies have used a method of attaching an organ of Corti on glass coverslips coated with &#946;-mercaptoethanol after explantation. Although the same method was used here, it was difficult to examine the tissues because they often detached or rolled up from the coverslip. Therefore, a new trial was performed to make it easier to monitor the tissue. First, an organ of Corti was placed on a plastic coverslip, and then the tissue was immobilized on the coverslip by pressing the remaining portion of the Reissner&#39;s membrane generated during the dissection using forceps.
This enabled the stable attachment of the organ of Corti to the coverslip so that an image could be acquired successfully by confocal microscopy. Although the tissue culture method presented here is an inexpensive and time-saving, development of proficiency in dissection does take some time. Known tissue culture methods include the attachment of the tissue by coating the coverslip with poly-L-ornithine and laminin16; applying organotypic cell culture inserts that do not require adhesive or coating materials17 thereby eliminating the need for adhesive substances, which reduces explant damage; and using a tissue adhesive composed of proteins extracted from marine mussels10. This protocol requires neither coating materials nor organotypic membranes nor tissue adhesives; an inexpensive plastic coverslip is sufficient. Better images can be acquired if an area of 8 mm x 8 mm is considered along with a Z-stack of the tissue thickness of the organ of Corti. Considering the vast amount of data to be stored and the appropriate time for time lapse, images of whole cell movement were acquired, focusing on the MSCs instead of the organ of Corti.
The results shown in Figure 4 seem to suggest that both the organ of Corti and MSCs can be visualized as clear video images when the images taken are 2 mm long, and the Z-stack is combined with a time lapse. Here, although a co-culture experiment was performed with MSCs and an explant, this setting could be applied to different cell types or conditions for future studies. For example, it will help to apply this protocol to examine the effects of drugs or exosomes that protect the damage of cochlea. Embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) can functionally and morphologically regenerate into mechanosensitive hair cell-like cells18. Therefore, this protocol can be applied to study the differentiation of iPSCs or ESCs into auditory hair cells or support cells. This ex vivo method can aid in regenerative medicine to restore hearing.

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.remotexs.ntu.edu.sg/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><tbody><tr><td>10X PBS Buffer</td><td>GenDEPOT</td><td>P2100-104</td><td></td></tr><tr><td>4% Formalin</td><td>T&amp;amp;I</td><td>BPP-9004</td><td></td></tr><tr><td>Ampicillin</td><td>sigma&amp;nbsp;</td><td>A5354-10ml</td><td></td></tr><tr><td>BSA</td><td>sigma&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 MSCs are transplanted into tissue composed of such complex cells, the use of technology to ascertain where cells are recruited and stabilized is pivotal to understand the mechanism of cell therapy and regeneration using MSCs. In addition, to determine how MSCs are localized in the presence or absence of damage, a 2D method such as a wound healing method is more suitable than a Transwell system in which cells move randomly downward in one direction.
In this study, the two-dimensional path of MSCs was tracked in a wound healing method by simply using a glass cylinder as a barrier between the MSCs and the organ of Corti and removing the cylinder after the MSCs are attached. When the organ of Corti was cultured, fibroblasts grew very quickly outward from the outer hair cells present in the outermost layer (Figure 3, Video 1). Therefore, most GFP-labeled MSCs seemed to be pushed out by fast-growing fibroblasts (Video 1). Nonetheless, some GFP-labeled MSCs penetrated the layer of fibroblasts and successfully migrated to the organ of Corti (Figure 3, Video 1).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61947/61947fig03.jpg" /> 
Figure 3. Confocal image of the organ of Corti and GFP-labeled MSCs. After the removal of the glass cylinder and an additional 4 h of incubation, fixation and staining were performed. Scale bar =100 &#181;m. Abbreviations: GFP = green fluorescent protein; MSCs: mesenchymal stem cells.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61947/61947fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1:&#160;Time-lapse confocal microscopy of the organ of Corti and GFP-labeled MSCs.&#160;After the removal of the glass cylinder and an additional 4 h of incubation, fixation and staining were performed. Scale bar =100 &#181;m. Abbreviations: GFP = green fluorescent protein; MSCs: mesenchymal stem cells.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61947/Video JoVE.mp4" target="_blank">Please click here to download this video.</a>
The results from 72 h of incubation showed that MSCs migrated to the organ of Corti and were mainly localized in the modiolus and the area where the cochlear nerve fibers separated from the modiolus were gathered; the morphology of the MSCs was altered to a radial shape similar to that of nerve fibers (Figure 4B). Intriguingly, cells were transformed into a linear shape along the limbus line rather than the hair cell area (Figure 4E, see arrow). Although the apical and basal ends of the organ of Corti were combined to prevent the MSCs from moving to the medial side of basilar membrane, MSCs were able to migrate and grow in the nerve fiber collection area by penetrating the various cell layers and physical barriers (Figure 4E). When damage to hair cells was induced by treatment with 1 mM kanamycin for 16 h, and cells were cultured for a further 72 h after the removal of kanamycin, MSCs were localized not only in the modiolus, but also in the outer hair cells (Figure 4C,D).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61947/61947fig4v2.jpg" /> 
Figure 4. Confocal images of the organ of Corti and GFP-labeled MSCs. (A) After the removal of the glass cylinder and an additional 16 h of incubation, an image was taken after fixation and staining. Scale bar =200 &#181;m. (B) MSCs were predominantly distributed in the modiolus area, scale bar =20 &#181;m. After the removal of the glass cylinder from the dish, the cells were further incubated with 1.0 mM kanamycin for 16 h, cultured with kanamycin-free medium for 72 h, and then fixed and stained. Images of the (C) outer hair cell region, scale bar= 20 &#181;m; (D) modiolus region, scale bar= 50 &#181;m; and (E) whole cochlea, scale bar= 100 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61947/61947fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
These results indicate that the experimental system demonstrated in this study would be a useful test tool for developing cell therapeutic strategies using MSCs and can be applied specifically to study hearing loss caused by various factors.

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 ...

in-vitro-time-lapse-live-cell-imaging-to-explore-cell-migration

Nanyang Technological University

Research

JoVE Journal

Medicine

3.7K Views.  Yonsei University Wonju College of Medicine. 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.

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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengineering

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Neuroscience

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Developmental Biology

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

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

Summary

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Chapters in this video

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons