2. Aspirate cell suspension in a 10 ml syringe.

3. Carefully fill up the tubes inside the bioreactor with the cell

suspension (see Note 5).

4. Place the bioreactor chamber on a roller mixer inside a cell

culture incubator at 37
C.

5. Rotate for a total time that is suitable for first attachment of the

used cells (e.g., in this setup, we rotated at 2.0 revolutions/

30 min).

6. In the meantime, seed the positive controls in 12 well plates in

triplicates, using the same normalized cell count as the cells

seeded on the grafts. The positive controls will be used to

determine the seeding efficiency as explained in Subheading

3.6.

7. Stop the rotation and incubate the bioreactor under static

conditions for the time that affirms safe attachment of the

used cells (in this setup, we chose 3.5 h) (see Note 6).

8. Subsequently, after 3.5 h of static cultivation, take the bioreac-

tor chamber under the sterile bench.

9. Discard the remaining suspension (see Note 7).

10. Gently flush once with PBS to remove non-adherent cells.

11. Replace all 3-way stopcocks by new sterile ones.

12. Attach the chamber to the perfusion setup.

13. Assemble all remaining syringes, 3-way-connectors and filters.

14. Fill up the perfusion setup with the required volume of media

for cultivation. Here, we used 50 ml of cell culture medium

containing 10% fetal calf serum and 1% penicillin/streptomycin

supplement.

15. Connect the system to the roller pump inside the perfusion

platform.

16. Start the perfusion and adjust flow conditions according to the

planned approach. Here, we used a flow rate of 1.5 ml/min

with pulsatile flow, 0.5 Hz.

17. Make sure all remaining air bubbles are flushed towards the

reservoir bottle.

18. Continue perfusion for the intended cultivation time. Here, we

applied a total length of 5 days (i.e., 96 h).

3.5

Lactate

Monitoring

An evaluation of lactate production was considered standard to

estimate the cell metabolism in tissue engineered constructs,

which is also commonly applied for the process accompanying

analytics in bioreactor assisted cultivations since the emergence of

tissue engineering field in the 1990s [15]. In particular, by a simple

batchwise media perfusion, it can be easily integrated by sequential

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