2

Materials

A bioreactor which can provide uniaxial stretching: Autoclave

(1.5 h at 134
C) the bioreactor culture chamber, sample hooks,

glass cover, and screws before use. Sterilize the motor module and

connection cable by ultraviolet rays (UV) exposure overnight.

2.1

Isolation of Mice

TDSCs

1. 6–8-week-old C57BL/6 mice.

2. T-25 cell culture flask.

3. Centrifuge.

4. 70% ethanol.

5. Phosphate buffer saline (PBS).

6. Sterile dissection kit for small animals.

7. Wash buffer: 100 U/mL penicillin and 100 μg/mL streptomy-

cin in PBS.

8. 3 mg/mL Type 1 collagenase.

9. 70 μm cell strainer.

10. Complete medium: Alpha Modified Eagle Medium supple-

mented with 10% fetal bovine serum (FBS), 100 U/mL peni-

cillin, 100 μg/mL streptomycin.

11. Fluorescence-activated Cell Sorting (FACS) buffer: PBS with

5% FBS.

12. Conjugated antibodies for flow cytometry: CD44, CD45,

CD90, and CD34.

2.2

Mechanical

Stimulation

2.2.1

Scaffold-Free

Mechanical Stimulation

1. TDSCs: Passage 2–4.

2. T-75 cell culture flask.

3. Stimulation medium: 25 ng/mL connective tissue growth fac-

tor and 4.4 mg/mL ascorbic acid in complete medium.

4. Complete medium.

5. PBS: prewarmed to 37
C.

6. Sterile tweezer.

7. Trypsin-EDTA solution: 0.25% trypsin and 0.02% EDTA.

8. 100  20 mm Petri dish.

2.2.2

Scaffold-Based

Mechanical Stimulation

1. TDSCs: Passage 2–4.

2. Complete medium.

3. 100  20 mm Petri dish.

4. CelGro^® collagen scaffold.

5. Customed culture chamber: 1.5 cm  2 cm, autoclave

before use.

3D Mechanical Stimulation to Tendon Stem Cell

137