5. Benchtop centrifuge with temperature control and swinging

bucket rotor e.g., Allegra X-12R, Beckmann Coulter, Brea

(CA), USA.

6. Roller mixer capable of slow rotation (here, we used 2 revolu-

tions/30 min).

7. Fluorescence microscope (EVOS Auto 2, ThermoFisher Scien-

tific, MA, USA).

8. Confocal microscope (Leica TCS SP8, Leica Microsystems

GmbH, Germany).

9. Multimode plate reader (such as Tecan Infinite^® 200 PRO).

3

Methods

3.1

General

Considerations

l

The presented method is used to determine the success of cell

homing after seeding in artificial small diameter vascular grafts

and subsequent evaluation of metabolic activity and preservation

of basic endothelial cell functions over a short period of 5 days.

l

The needed solutions should be prepared freshly on the day of

their usage.

l

All autoclavable parts should be autoclaved in advance to allow

them to cool down before usage on the first day of the

experiments.

l

The cells used for the experiments were harvested on the day of

cell seeding, thus cultivation has to be started 3–5 days in

advance, depending on the cell type.

l

In our experiments we used cells cultivated under static condi-

tions as positive controls to determine the seeding efficiency on

the grafts. Therefore, the cells were seeded out on day 1 of the

experiment in a 12-well plate with the same density compared to

cells seeded on the luminal side of the vascular grafts and

cultivated for 24 h until the first WST-1 assay.

3.2

Bioreactor Setup

Prior to conducting any experiments, set up the bioreactor system

as shown in Fig. 1 in an unsterile fashion to make sure that all parts

are fitting and to practice the handling. Adjust the length of the

silicon tubes to ensure that the system fits inside the bioreactor

perfusion platform. When the setup has been established, autoclave

the silicone tubings with the respective Luer Lock connectors, the

medium reservoir bottles and the pump tubing. Disconnect the

bioreactor and autoclave separately. Do not autoclave syringes,

3-way stopcocks and filters, as these are intended for single use

and not autoclavable.

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Max Wacker et al.