4

Notes

1. Ensure complete separation of tendons, and avoid any residual

muscles or other tissues. Otherwise, it may affect the purity of

the TDSCs finally obtained.

2. Vortex 10 s per 30 min when digest.

3. Keep cells on ice at all times for the following staining

procedure.

4. It is important to completely resuspend it. Evenly distributed

cell is one of the keys to form a quality cell sheet.

5. Change the medium every 3 days.

6. Change the medium every 3 days. Operation when changing

medium must be gentle. The medium flow should not directly

touch the cells as it could tear the cell sheet.

7. Extracellular matrix will become thick and present to be cloudy

when observed from the bottom of the flask after stimulated by

stimulation medium, which means the cell sheet is sufficiently

generated. Ideally, 6 days should be enough to observe the

margin of the cell sheet is rolled up. Rolled edge is the signal

to move to the next step.

8. Culture overnight at 37
C with 5% CO2 before stretching can

shrink the edge of the sample and more stably form the 3D

construct.

9. Add medium into the chamber firstly to ensure firmly assem-

bled and no leaking for the culture chamber.

10. Washing collagen-tenocyte construct should be gentler than

washing scaffold-free tendon-like 3D construct and do not

unfold the construct when washing the collagen-tenocyte

construct.

Table 1

Primer sequences used for RT-qPCR analysis

Gene

Primer sequence

Forward 5⁰ ! 3⁰

Reverse 5⁰ ! 3⁰

COL1A1

TGACTGGAAGAGCGGAGAGT

GTTCGGGCTGATGTACCAGT

Scleraxis

CCCAAACAGATCTGCACCTT

GGCTCTCCGTGACTCTTCAG

Mohawk

GTCCGGCAGCCAGATTTAAG

TCGCTGAGCTTTCCCCTTTA

Tenomodulin

CCGCAGAAAAGCCTATTGAA

GACCACCCATTGCTCATTCT

36B4

CTTCCCACTTGCTGAAAAGG

CGAAGAGACCGAATCCCATA

142

Ziming Chen et al.