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).

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
<ol>
	<li>Make proliferation medium for culture of PDL cells: &#945;-MEM medium supplemented with 10% FCS and 100 U/mL pen/strep.</li>
	<li>Prepare a container with ice to transfer isolated third molars.</li>
	<li>Sterilize surgical instruments by using an autoclave.</li>
	<li>Extract normal human impacted thir...

<ol>
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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...

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:718px;" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">&#945;-MEM</td>
			<td style="width:158px;">Gibco</td>
			<td style="width:166px;">11900-073</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">acetic acid&#160;</td>
			<td style="width:158px;">Sigma-Aldrich</td>
			<td style="width:166px;">64197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture flask 25 cm2</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture flask 75 cm2</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">430641</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:223px;">Chitosan</td>
			<td style="width:158px;">Heppe Medical&#160;Chitosan GmbH</td>
			<td style="width:166px;">/</td>
			<td>molecular weight 500 kDa, degree of deacetylation 85%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCS</td>
			<td style="width:158px;">Gibco</td>
			<td style="width:166px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td style="width:158px;">Molecular&#160;Probes</td>
			<td style="width:166px;">L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaOH</td>
			<td style="width:158px;">Sigma-Aldrich</td>
			<td style="width:166px;">1310732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">PBS</td>
			<td>KeyGen Biotech&#160;</td>
			<td style="width:166px;">KGB5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">pen/strep</td>
			<td style="width:158px;">Gibco</td>
			<td style="width:166px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trypsin/EDTA&#160;</td>
			<td>KeyGen Biotech&#160;</td>
			<td style="width:166px;">KGM25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15 mL conical centrifuge tube</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">24-well plate</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">3524</td>
			<td></td>
		</tr>
	</tbody>
</table>

formation of human periodontal ligament cell spheroids on chitosan films

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.

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...

Watch this Scientific Journal Video about Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films at JoVE.com

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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) ...

This is a detailed protocol of culturing periodontal ligament cell spheroids by chitosan films. Compared with the conventional culture system, the spheroid culture system produces periodontal ligament cells with increased self-renewal and osteogenic differentiation abilities. To begin, prepare 1%weight-to-volume chitosan solution as directed in the manuscript.Add 0.5 milliliters of chitosan solution into each well of a 24-well tissue culture polystyrene plate. Dry the plate in an oven at 60 degrees Celsius for 24 hours to form chitosan films. To neutralize the chitosan films, add 0.5 milliliters of 0.5 normal sodium hydroxide to each well of the 24-well plate and incubate at room temperature for two hours.Then, wash the chitosan films three times with double-distilled water. Sterilize the chitosan-coated 24-well plate in 70%alcohol overnight at room temperature. Rinse the plate three times with PBS then leave it under ultraviolet light overnight.Prior to cell seeding, pre-warm proliferation medium, PBS solution, and trypsin EDTA solution to 37 degrees Celsius. Discard the supernatant from the cell culture flask and wash the confluent periodontal ligament, or PDL cells, with 10 milliliters of PBS. Discard the PBS and add one milliliter of trypsin EDTA solution to the flask.Incubate the flask at 37 degrees Celsius for three minutes and then add three milliliters of proliferation medium to terminate the trypsin digestion. Transfer the cell suspension to a 15 milliliter conical tube then centrifuge it for five minutes at 300 times g. Discard the supernatant and re-suspend the cells in 500 microliters of proliferation medium.Count the cells with a hemocytometer and seed them onto the plate at five, 10, 30, and 60 thousand cells per square centimeter. Culture the PDL cells in an incubator at 37 degrees Celsius in 5%carbon dioxide and humidified air, changing the culture medium twice per week. Observe cell morphology daily for five days via an inverse phase contrast microscope.Viable PDL cell spheroids are successfully formed using this protocol. Spheroids are rarely observed for the lower-seeding density on days one and three. But at the two higher densities, various sizes of spheroids are present from day one.The optimal seeding density is 30 thousand cells per centimeter squared because it results in homogeneous spheroids. After five days of culture, larger spheroids are observed for all seeding densities. A viability assay demonstrates that the majority of PDL cells are alive on days one, three, and six, but the number of dead cells increases on day six.One limitation of this protocol is the decreased proliferation of cells after spheroid formation. So further studies to promote the proliferation of cells after spheroid formation are required.

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JoVE Journal

Developmental Biology

5.3K visualizações.  Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration. Este é um protocolo detalhado de cultivo de esferoides de células ligamentares periodontais por filmes quitosanos. Comparado com o sistema de cultura convencional, o sistema de cultura esferoide produz células do ligamento periodontal com maior autoconexão e capacidades de diferenciação osteogênica. Para começar, prepare a solução chitosana de peso para volume de 1%, conforme indicado no manuscrito.Adicione 0,5 mililitros de solução de chitosan em cada poço de uma placa de...