4
C. Wash cells after incubation with ice-cold PBS for three

times.

8. Add secondary antibodies to the samples in 2 μg/mL and

incubate for 30 min at 4
C in dark. Wash samples again for

three times with PBS and resuspend by ice-cold FACS buffer.

The samples were then analyzed by flow cytometry within 1 h.

3.2

Mechanical

Stimulation

3.2.1

Scaffold-Free

Mechanical Stimulation

1. Add 8 mL complete medium to the warmed-up cells (mice

TDSCs, one million cells, passage 2–4, to 37
C). And use a

pipette to transfer cells to a 15 mL centrifuge tube. Centrifuge

at 350  g for 5 min.

2. Decant the medium, and resuspend cells gently in 1–2 mL of

complete medium. Gently pipette up and down for several

times to completely resuspend it (see Note 4).

3. Transfer resuspended cells to a T-75 flask. Add complete

medium to the flask to reach a total volume of 10 mL. Place

the flask into the incubator and culture at 37
C with 5% CO2.

Cell culture should be done until the cells are cultured to 100%

confluence (see Note 5).

4. Discard the complete medium. Add 10 mL of stimulation

medium slowly and avoid touching the cells attached to the

bottom of the flask.

5. Culture the cells in stimulation medium for 6 days (see Note 6)

to sufficiently generate the cell sheet at 37
C with 5% CO2 (see

Note 7).

6. Discard the stimulation medium completely, and wash the

monolayer cell sheet with prewarmed PBS by swirling the

flask. Then discard the PBS. Use a pipette to add 1 mL of

trypsin-EDTA solution to the corner of the flask.

7. Gently tap the corner of the flask to detach the cell sheet until

the corner of the cell sheet starts to peel off from the bottom of

the flask (see Fig. 2a).

8. Add 9 mL of complete medium to stop the trypsinization.

Keep swirling the flask to completely peel off the cell sheet.

9. Pour the whole cell sheet into a Petri dish with medium. Use a

sterile tweezer to pick up one corner of the cell sheet and rotate

in a clockwise direction for 15 times. Then pick up another end

of the cell sheet and rotate in an anti-clockwise direction for ten

times to firmly generate a tendon-like 3D construct (see

Fig. 2a).

10. Connect the hooks by the connecter and adjust the distance

between two hooks to 2 cm. Gently wind the 3D TDSCs

construct on the assembled hook for three times on each hook.

3D Mechanical Stimulation to Tendon Stem Cell

139