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.
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.
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 previous systematic review revealed that PDL cells showed great potential for periodontal regeneration5. 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 culture6. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment7. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells8. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture.
Conventional 3D culture method is encapsulating cells in 3D biomaterials. Compared with cells encapsulated in 3D biomaterials, cellular spheroids mimic the in vivo situation more closely because spheroids are aggregates of cells growing free of foreign materials9,10,11,12. It is reported that cellular spheroids promoted MSC bioactivities via the preservation of extracellular matrix (ECM) components including fibronectin and laminin13. To improve conventional PDL cell culture models, we have recently developed a 3D PDL cell culture method, which is based on spheroid formation of PDL cells on chitosan films14. Spheroid formation increased the self-renewal and osteogenic differentiation capacities of PDL cells14. Here, we present detailed PDL cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the shortcomings related to conventional TCPS cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.
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
2. Preparation of chitosan films
3. Cell seeding
4. Cell survival
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/cm2, various sizes of PDL cell spheroids were found since day 1. PDL cell spheroid formation was observed from all the seeding densities after 3 days. As shown in Figure 1, 3 x 104 cells/cm2 was the optimal PDL cell seeding density because the size of PDL cell spheroids was homogeneous at this cell seeding density. After 5 days of culture, larger spheroids were formed for all the cell seeding densities. Suspended PDL cells were rarely found on day 5.
The viability of PDL cells in spheroids was assessed after 1, 3 and 6 days. As shown in Figure 2, the majority of cells in spheroids were living cells on day 1, 3, and 6. While on day 6, the number of dead cells was increased in the central part.
Figure 1. The morphology of PDL cellular spheroids.
For the seeding density of 0.5 x 104 cells/cm2, PDL cell spheroids were rarely observed on day 1 and 3. For the seeding densities of 3 x 104 and 6 x 104 cells/cm2, various sizes of PDL cell spheroids were found since day 1. PDL cell spheroid formation was observed from all the seeding densities after 3 days. After 5 days of culture, larger spheroids were formed for all the cell seeding densities. Suspended PDL cells were rarely found on day 5. Scale bar: 200 μm. This figure has been modified from Yan et al.14. Please click here to view a larger version of this figure.
Figure 2. The viability of PDL cellular spheroids on chitosan films.
The viability of PDL cells in spheroids was assessed after 1, 3 and 6 days. As shown here, the majority of cells in spheroids were living cells on day 1, 3, and 6. While on day 6, the number of dead cells was increased in the central part. Scale bars: 200 μm. This figure has been modified from Yan et al.14. Please click here to view a larger version of this figure.
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 simply pipetting the medium a few times14. Thus, ECM and intercellular junctions can be well preserved.
The critical steps of this protocol include: (1) making sure that the surgical instruments and chitosan films are sterilized for cell culture; (2) scraping the PDL from the middle third part of the root to avoid contaminations from gingival or apical tissues; and (3) requiring higher cell seeding densities (≥3 x 104 cells/cm2) for the successful spheroid formation of PDL cells.
However, one limitation for this method is that decreased proliferation of PDL cells was observed after spheroid formation14, which is similar to some previous studies that performed on adipose stromal cells10 and hepatocellular carcinoma cell line15. This is probably caused by the difference in the regulation of cyclin-dependent kinase inhibitors between spheroid and monolayer cells16. To benefit clinical application, further studies to promote the proliferation of cell spheroids are required.
Another drawback of the cellular spheroid is the inadequate diffusion in the core. Although PDL cells were mainly alive in spheroid culture, the number of dead cells was increased in the central part of spheroids on day 6. This phenomenon was probably because the diffusion of oxygen and nutrients is compromised in the spheroid core as PDL cell spheroids become larger17,18.
Other methods that reported to induce cellular spheroids include a non-adherent surface19, microwells20, spinner flasks21, micropatterned surfaces22,23, hanging drops24, and a forced-aggregation technique25. Compared to aforementioned methods, chitosan films are relatively cost-effective and easy to reproduce. Another advantage of this method is the antimicrobial properties of chitosan26, which is significantly beneficial for in vitro cell culture.
In summary, here we present detailed 3D cell spheroid culture protocols based on chitosan films. The culture of PDL cellular spheroids overcome some of the limitations related to conventional 2D TCPS cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.
The authors have nothing to disclose.
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).
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 |