3.3

Bioreactor Setup

for De-epithelialization

1. Put on gloves, remove everything from the BSC, and disinfect

it with 70% ethanol.

2. Disinfect tools, DMEM + Antibiotics, detergents, deionized

filtered H2O, PBS, and other materials one by one (see Note 6).

3.3.1

Detergent

Circuitries

1. Label the Erlenmeyer flasks with a marker, and fill each flask

with the respective solution (a, b, c, or d) using one plastic

pipette per flask as follows:

(a)

Add 250 mL of H2O to the 250 mL flask.

(b)

Add 140 mL of Triton X-100 to a 125 mL flask.

(c)

Add 140 mL of PBS to a 125 mL flask.

(d)

Add 75 mL of SDS to a 125 mL flask.

2. Insert two tubing into each rubber stopper as follows (see

Note 7):

(a)

H2O flask: Place one 5 cm tubing in one stopper hole, and

one 25 cm tubing in the other hole down to the bottom of

the flask.

(b)

Triton X-100 flask: Place one 5 cm tubing in one stopper

hole, and one 15 cm tubing in the other hole down to the

bottom of the flask.

(c)

PBS flask: Place one 5 cm tubing in one stopper hole, and

one 15 cm tubing in the other hole down to the bottom of

the flask.

(d)

SDS flask: Place two 25 cm tubing in each stopper hole,

both down to the bottom of the flask.

3. Introduce each stopper into the corresponding flask.

4. Attach one Tee connector to each of the following: H2O short

tubing, both Triton X-100 tubing, and PBS long tubing

(Fig. 2).

5. Attach one elbow connector to each of the following: H2O

long tubing, PBS short tubing, and both SDS tubing.

6. Connect both H2O and Triton X-100 flask short tubing using

a 15 cm tubing.

7. Connect the Triton X-100 and PBS flask short tubing using a

15 cm tubing.

8. Connect both H2O and Triton X-100 flask long tubing using a

15 cm tubing.

9. Connect the Triton X-100 and PBS flask long tubing using a

15 cm tubing.

10. Attach a 15 cm tubing to H2O short tubing tee connector.

Then, connect it to a one-way valve (flow direction towards the

H2O flask).

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