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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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'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 ....

Protocol

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

  1. 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.
  2. Place dissection instruments in 70% ethanol for 10 min,....

Representative Results

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's cells, outer hair cells, inner hair cells, nerve fibers, basilar membrane, and reticular lamina12. When M.......

Discussion

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. applied MSCs to human beings trans-venously, they did not achieve any significant improvement in hearing14. Until recently, nearly 12 expe.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
10X PBS BufferGenDEPOTP2100-104
4% FormalinT&IBPP-9004
Ampicillinsigma A5354-10ml
BSAsigma A4503-100G
confocal dishSPL200350
confocal microscope ZEISSLSM800
coverslipSPL20009
DMEM/F12Gibco10565-018
Fetal Bovine SerumThermo Fisher scientific16140071
Fluorsheild with DAPIsigma F6057
ForcepDumont0508-L5-P0
HBSSThermo Fisher scientific14065056
HEPESThermo Fisher scientific15630080
N2 supplementGibco17502-048
Phalloidin-iFluor 647 Reagentabcamab176759
Stage Top IncubatorTOKAI HITWELSX
Strain C57BL/6 mouse messenchymal stem cells with GFPcyagenMUBMX-01101
Triton X-100sigma T8787

References

  1. Brown, C. S., Emmett, S. D., Robler, S. K., Tucci, D. L. Global hearing loss prevention. Otolaryngologic Clinics of North America. 51 (3), 575-592 (2018).
  2. Chamberlain, G., Fox, J., Ashton, B., Middleton, J.

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In VitroTime lapseLive cell ImagingCell MigrationOrgan Of CortiMesenchymal Stem Cells MSCsTranswellWound HealingTectorial MembraneCochleaOrgan CultureTissue Dissection

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