or contact inhibition before the planned termination of the

experiment.

5. To seed the cells, hold the bioreactor in an upright position.

Attach the syringe at the lower 3-way stopcock which is now

attached to the grafts at the bottom of the reactor chamber.

Carefully fill the tubes until the cell suspension reaches the

upper 3-way stopcocks attached to the tubes on top of the

bioreactor. Close all 3-way stopcocks.

6. To determine the optimal timing for initial attachment of the

cell type used in the experiments, it is advisable to test the time

required for attachment in advance by 2D seeding experiments

in well plates under optimal conditions, e.g., coatings that are

recommended for the respective cell type.

7. Attach two syringes to the 3-way stopcocks at both sides of

chamber. One syringe must be empty, the other should be filled

with sterile air. Hold the chamber in an upright position again,

with the air-filled syringe attached to the top of the chamber.

Flush out the seeding medium while replacing it with air out of

the upper syringe. Replace both syringes. This time one syringe

should be filled with warm PBS. Flush 2 ml of warm PBS

through each tube. Collect perfused PBS with an empty

syringe on the other side. If necessary, the washout can be

stored for further analysis of the non-adherent cells or e.g.,

cell count from washout, which can provide first insights to the

number of adherent cells.

8. As cell metabolism differs between cell lines and high concen-

tration of WST-1 assay might impact cell viability, the optimal

concentration of WST-1 assay solution should be tested in

advance under static conditions to evaluate the lowest concen-

tration that still allows detecting cell proliferation.

9. If not described otherwise, all staining procedures of tube

sections were carried out in 2 ml microreaction tubes.

10. If not described otherwise, all washing steps were performed

for 5 min at room temperature.

Acknowledgments

This project was funded by the German Research Foundation

(grant number WA 4489/1-1).

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Max Wacker et al.