Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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 into the proliferation medium and transfer into the laboratory at 4 °C.
6. Place the extracted tooth in a sterile 100 mm Petri dish, working within a biohazard laminar flow hood.
7. Add 10 mL of PBS to wash the extracted tooth. Repeat the washing step twice.
8. By using a sterile scalpel, gently separate the PDL from the root.
   NOTE: PDL should be scraped from the middle third part of the root to avoid contaminations from gingival or apical tissues.
9. Mince PDL into 1-2-mm fragments with the help of a sterilized scalpel blade.
10. Place PDL fragments in a T-25 culture flask, filled with 3-4 mL of proliferation medium.
11. Culture the samples in an incubator at 37 °C in 5% CO₂ with humidified air.
12. Change the culture medium after outgrowth of PDL cells was observed. It usually takes 1-2 weeks for the outgrowth of PDL cells. Observe the outgrowth of PDL cells via an inverse phase contrast microscope.
13. Change the culture medium twice per week thereafter.
14. When PDL cells have reached confluence, sub-culture cells at ratio of 1:4 (passage 1).
15. Observe cell growth daily and change the culture medium twice per week.
16. Perform each subsequent passage at ratio of 1:4 after the cells achieve 80% confluence. Use third- to fifth-generation of PDL cells for cell seeding.

2. Preparation of chitosan films

1. For the preparation of a 1% (v/v) acetic acid solution, add 5 mL of acetic acid to 495 mL of double-distilled water in a clean glass beaker.
2. For the preparation of 1% w/v chitosan solution, weigh 5 g of chitosan powder (average molecular weight 400 kDa, 85% degree of acetylation) and add to 495 mL of 1% (v/v) acetic acid solution.
3. Stir the prepared solution for 24 hours with stirring bar and magnetic stirring plate at 60 °C.
4. Autoclave the prepared solution to ensure complete dissolution of chitosan.
   NOTE: The experiment can be paused at this stage.
5. To form chitosan films, add 1% w/v chitosan solution into tissue culture polystyrene (TCPS) dishes at 0.25 mL/cm². For example, add 0.5 mL of 1% w/v chitosan solution to one well of 24 –well plate.
6. Dry TCPS dishes in an oven at 60 °C for 24 hours to form chitosan films.
7. Prepare 0.5 N sodium hydroxide (NaOH). Stir 10 g of NaOH, a little at a time, into a large volume of double-distilled water by using the magnetic stir bar, and then dilute the solution to make 0.5 L.
   NOTE: Add NaOH to water–do not add water to solid NaOH. Do not touch NaOH! It could cause chemical burns.
8. Neutralize chitosan films with 0.5 N NaOH. Add 0.5 mL of 0.5 N NaOH solution to one well of 24-well plate for 2 hours.
9. Wash chitosan films three times with double-distilled water.
   NOTE: The experiment can be paused at this stage.
10. Prior to cell seeding, sterilize the chitosan-coated 24-well plates in 70% alcohol overnight at room temperature.
11. Rinse chitosan-coated plates three times with PBS at room temperature.
12. Sterilize chitosan-coated plates by treatment under ultraviolet light overnight at room temperature.

3. Cell seeding

1. Pre-warm proliferation medium PBS solution and trypsin/EDTA solution to 37 °C.
2. Discard the supernatant from the T-75 cell culture flask.
3. Wash confluent PDL cells with 10 mL of PBS solution.
4. Discard the PBS solution.
5. Add 1 mL of trypsin/EDTA solution to T-75 cell culture flask.
6. Incubate cells with trypsin/EDTA solution for 3 min at 37 °C.
7. Terminate the digestion process of trypsin by adding 3 mL of proliferation medium to T-75 cell culture flask.
8. Transfer the prepared cell suspension into a 15 mL conical centrifuge tube.
9. Centrifuge the suspension for 5 min at 300 x g and room temperature.
10. Discard the supernatant from the 15 mL conical centrifuge tube and re-suspend the cell pellet in 500 µL of proliferation medium.
11. Count PDL cells with a hemocytometer.
12. Seed PDL cells at the densities of 0.5 x 10⁴, 1 x 10⁴, 3 x 10⁴, and 6 x 10⁴ cells/cm².
13. Culture PDL cells on chitosan films in an incubator at 37 °C in 5% CO₂ with humidified air.
14. Change the culture medium twice per week thereafter.
15. On days 1, 2, 3, 4, and 5, observe the cell morphology daily via an inverse phase contrast microscope.

4. Cell survival

1. After 1, 3, and 6 days of culture, determine the survival of cells using a commercial viability/cytotoxicity kit.
2. In a 15 mL tube, prepare 2 mL of a fresh solution of 2 mM calcein-AM and 4 mM ethidium homodimer in PBS by vortexing for 5 s.
   NOTE: Keep the process in the dark.
3. Remove the culture medium and wash spheroids in PBS.
4. Incubate spheroids in of PBS containing 2 mM calcein-AM and 4 mM ethidium homodimer for 30 min at room temperature in the dark.
5. Rinse spheroids twice in PBS. For spheroids from one well of 24-well plate, use 2 mL of PBS for each time.
6. Observe the samples by using a fluorescence microscope using 10x or 20x magnification.