As it is essential to maintain sterility, all following steps need to

be carried out under a sterile cell culture bench (Laminar air flow).

Wear sterile, surgical gloves and gown during experiments. Addi-

tionally, we spread out a sterile surgical sheet inside the cell culture

bench.

3.3

Connection of

Small Diameter

Vascular Grafts to

Bioreactor Chamber

1. Open the autoclaved bioreactor chamber.

2. Pull the first graft over the barb connectors inside the bioreac-

tor chamber using tweezers.

3. Use non resorbable surgical sutures to tie in the tube (see

Note 3).

4. Attach one 3-way stopcock to each graft outlet of the bioreac-

tor chamber.

5. Attach a syringe filled with 5 ml of PBS to one 3-way stopcock.

6. Carefully fill tube with PBS, holding the bioreactor chamber in

an upright position with the syringe connected to the bottom

3-way stopcock to allow the air to evacuate. Determine the

volume needed to completely fill the lumen between the two

3-way stopcocks upstream and downstream of the graft with

fluid. This is the volume needed for endothelial cell seeding and

WST-1 assay.

7. Close the 3-way stopcock on the opposite side.

8. Carefully apply some pressure in order to check for leakage.

9. Repeat with the second graft following steps 2–8.

10. Close all open Luer Lock ports with sterile sealing cabs, close

the bioreactor chamber and store it in an incubator until seed-

ing of endothelial cells.

3.4

Cell Seeding

On the first day of the experiments, the endothelial cells of the

respective endothelial cell line will be seeded to the luminal surface

of the grafts. In advance, the cells should be pre-cultivated to a

confluency level of about 80% before the commencement of exper-

iment and harvesting for cell seeding. They can be harvested fol-

lowing standard cell culture protocols.

1. Prepare endothelial the cell suspension for seeding in a required

concentration. Adjust the working concentration to a cell num-

ber per luminal surface area that fits your testing hypothesis as

well as corresponding targeted cultivation time. It should be

noted that the same normalized cell count should also be

applicable to your static 2D controls (see Note 4). In our

specific setup we needed 3 ml of volume for each graft and

seeded with a density of 5  10⁴ cells per cm², thus the

concentration of the cell suspension for seeding was set to

4  10⁵ cells per ml.

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