6. Assemble and switch the bioreactor on as described in Sub-

heading 3.2.1, step 12.

3.3

Extraction of RNA

for Validation of the

System

1. Detach the sample from bioreactor by two clean tweezers, and

then wash the sample for three times by PBS (see Note 10).

2. Dissect a sample of 1 cm in the middle by a clean scissor (see

Note 11).

3. Snap-freeze the sample in liquid nitrogen.

4. Quickly

move

the

sample

into

a

clean

mortar

for

homogenization.

5. Add 1 mL TRIzol reagent into the mortar.

6. Vertically press the sample with a rocking motion to homoge-

nize the sample by a pestle, and then keep grinding the tissue

for about 5 min (see Note 12).

7. Transfer the homogenized samples into a 2 mL tube and

incubate for 10 min at room temperature.

8. Add 200 μL chloroform into the tube. Shake the tube vigor-

ously for 15 s by hand. And then incubate the sample for 3 min

at room temperature.

9. Centrifuge for 15 min at 4
C, 12,000  g. Transfer the

colorless upper aqueous phase into a clean 2 mL tube (see

Note 13).

10. Add same volume 70% ethanol to the aqueous phase. Vortex to

mix well and then spin down.

11. Transfer up to 700 μL of sample to the spin cartridge with the

collection tube. And then centrifuge at 12,000  g for 15 s at

room temperature. Discard the liquid in collection tube and

reinsert the spin cartridge. Repeat this step until all the samples

are processed (see Note 14).

12. Add 700 μL Wash Buffer I, and then centrifuge at 12,000  g

for 15 s. Insert the spin cartridge into a new collection tube.

13. Add 500 μL Wash Buffer II, and then centrifuge at 12,000  g

for 15 s. Discard the liquid in collection tube. Repeat this

step once.

14. Centrifuge at 12,000  g for 2 min to dry the membrane.

Insert the spin cartridge into a recovery tube. Add 30 μL

RNase-free water to the center of the spin cartridge. Incubate

for 1 min (see Note 15).

15. Centrifuge the spin cartridge for 2 min at 12,000  g. Now the

RNA is in the recovery tube and ready for RT-qPCR (Table 1).

3D Mechanical Stimulation to Tendon Stem Cell

141