Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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 treatments^2,3,4.

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 third molars from adults (18-28 years of age) at the Dental Clinic of School and Hospital of Stomatology, Tongji University.
5. Place third molars ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
α-MEM	Gibco	11900-073
acetic acid	Sigma-Aldrich	64197
Cell culture flask 25 cm2	Corning	430639
Cell culture flask 75 cm2	Corning	430641
Chitosan	Heppe Medical Chitosan GmbH	/	molecular weight 500 kDa, degree of deacetylation 85%
FCS	Gibco	26140-079
Live/Dead Viability/Cytotoxicity Kit	Molecular Probes	L3224
NaOH	Sigma-Aldrich	1310732
PBS	KeyGen Biotech	KGB5001
pen/strep	Gibco	15140-122
Trypsin/EDTA	KeyGen Biotech	KGM25200
15 mL conical centrifuge tube	Corning	430790
24-well plate	Corning	3524