media sampling. As the metabolism of endothelial cells mainly relies

on glycolysis and consequent lactate production [16], lactate was

measured in the cell culture medium in order to gain insight into

metabolic activity of seeded cells. Henceforth, 1 ml of cell culture

medium is collected through the syringes attached to the medium

reservoir, to measure the lactate concentration everyday.

1. Start with the first medium collection 30 min after starting

media perfusion.

2. Take up 1 ml of media into the last 10 ml syringe connected to

the media reservoir.

3. Close the corresponding 3-way stopcock so that the syringe is

disengaged from the medium reservoir.

4. Flush 5 ml of air from the second last syringe connected to the

media reservoir through the tubing inside the medium reser-

voir. This is done to ensure that no media is left in the

sampling tube.

5. Disconnect the last syringe with the medium and store the

sample at 80
C.

6. Repeat steps 2–5 each day of the experiment.

7. After completion of the experiment, thaw the media samples

and measure the lactate levels. In this setup, a large scale

analyzer was used for medical laboratories in collaboration

with the local institute for clinical chemistry. Alternatively,

e.g., colorimetric or fluorometric assays are commercially

available.

8. The acquired concentration was converted into an absolute

molar mass in consideration of the media volume at the time

of sampling. Exemplary results are shown in Fig. 2.

3.6

WST-1

Proliferation Assay

In recent past, WST-1 proliferation assay has been applied to quan-

titatively assess the cell proliferation in perfused tissue engineered

constructs [17–19]. Another beneficial application comes by the

ease to determine the seeding efficiency in the 3D artificial grafts in

direct comparison to the optimal conditions of vertical sedimenta-

tion onto a two-dimensional cell culture surface in the positive

controls. Especially as low efficiency rates for adhesion of seeded

cells seems to be a problem in colonization of 3D scaffolds [20], we

prefer a direct evaluation of the resulting vitalization over more

laborious cell counting.

We could see from preliminary experiments, that almost 100%

of cells would attach and proliferate when seeded in a well plate.

Therefore, the estimated seeding efficiency will be used as a mea-

sure for a ratio of cells that could attach to the tested grafts after

24 h relative to the positive controls in the wells.

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