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In This Article

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

Summary

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Abstract

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Introduction

Periodontitis, initialized principally by dental plaque1, is characterized by the damage of periodontal tissues including periodontal ligament (PDL), alveolar bone, and cementum. Current treatments for periodontitis are usually successful in preventing the progress of the active disease, but the regeneration of lost periodontal tissues remains a clinical challenge. Recently, important progress has been made in cell-based approaches for periodontal tissue regeneration to overcome the drawbacks of current treatments2,3,4.

Our ....

Protocol

The study protocol was approved by the Ethics Committee of School and Hospital of Stomatology, Tongji University. All patients provided written informed consent.

1. PDL cell isolation

  1. Make proliferation medium for culture of PDL cells: α-MEM medium supplemented with 10% FCS and 100 U/mL pen/strep.
  2. Prepare a container with ice to transfer isolated third molars.
  3. Sterilize surgical instruments by using an autoclave.
  4. Extract normal human impacted thir.......

Representative Results

Using the present protocol, viable PDL cell spheroids were successfully formed. Figure 1 showed that suspended cells or spheroids instead of attached cells were mainly observed on chitosan films. For the seeding density of 0.5 x 104 cells/cm2, attached PDL cells were occasionally found on day 1 and 3, and PDL cell spheroids were rarely observed. On the contrary, for the seeding densities of 3 x 104 and 6 x 104 cells.......

Discussion

The present study introduced a 3D cell culture system to overcome some limitations related to conventional 2D monolayer cell culture. According to the protocol, PDL cellular spheroids were successfully formed by culturing cells on chitosan films. Our previous study reported that spheroid formation increased the self-renewal and osteogenic differentiation capacities of PDL cells14. Instead of using an enzyme to harvest cells from TCPS, PDL cell spheroids could be harvested from chitosan films by si.......

Acknowledgements

This study was sponsored by National Natural Science Foundation of China (NSFC 81700978), Fundamental Research Funds for the Central Universities (1504219050), Natural Science Foundation of Shanghai (17ZR1432800), and Shanghai Medical Exploration Project (17411972600).

....

Materials

NameCompanyCatalog NumberComments
α-MEMGibco11900-073
acetic acid Sigma-Aldrich64197
Cell culture flask 25 cm2Corning430639
Cell culture flask 75 cm2Corning430641
ChitosanHeppe Medical Chitosan GmbH/molecular weight 500 kDa, degree of deacetylation 85%
FCSGibco26140-079
Live/Dead Viability/Cytotoxicity KitMolecular ProbesL3224
NaOHSigma-Aldrich1310732
PBSKeyGen Biotech KGB5001
pen/strepGibco15140-122
Trypsin/EDTA KeyGen Biotech KGM25200
15 mL conical centrifuge tubeCorning430790
24-well plateCorning3524

References

  1. Albandar, J. M. Epidemiology and risk factors of periodontal diseases. Dental Clinics of North America. 49 (3), 517-532 (2005).
  2. Bartold, P. M., McCulloch, C. A., Narayanan, A. S., Pitaru, S. Tissue e....

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Periodontal Ligament CellsCell SpheroidsChitosan FilmsCell CultureOsteogenic DifferentiationCell Seeding DensityCell Morphology

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