Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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.