3.7

Termination of

Experiment

1. After 96 h of cultivation, stop the perfusion, take out the grafts

and divide them into five equal samples by equidistant cross-

sectional cutting in order to perform the immune stainings and

AcLDL assay.

2. Store the graft sections in PBS and immediately proceed with

the assay and staining procedures.

3. The samples which are not used for AcLDL uptake assay should

be washed in PBS (in 50 ml falcon tube) for 5 min, followed by

fixation with in 4% PFA for 5 min (in 15 ml Ficoll tube). Wash

them twice afterwards in an excess volume of PBS in a 50 ml

falcon tube.

3.8

AcLDL Uptake

Assay (Modified from

Manufacturer’s

Instructions)

The AcLDL assay is an easy and direct method to verify the intact

functionality of the seeded endothelial cells by the uptake of acety-

lated low density lipoprotein (LDL) particles via scavenger recep-

tors expressed on an functionality intact endothelial cell [21]. As a

result, positive signals would indicate an unimpaired function of the

endothelial cells, while the lack of LDL signals would indicate

either impaired and degenerated endothelial cells or might repre-

sent a possible overgrowth by non-endothelial cells.

1. Place

one

of

the

samples

from

3.7.2

in

a

2

ml

microreaction tube.

2. Wash the designated sample two times with PBS+.

3. Cover the sample in an excess of AcLDL working solution. The

working solution consists of the respective cell culture medium

with 10% FCS and 1.25% (v/v) AcLDL supplied stock solu-

tion. For 1 cm of sample, we used 400 μl of AcLDL working

solution.

4. Incubate for 3.5 h at 37
C in a cell culture incubator.

5. Add one drop of NucBlue reagent.

6. Incubate for another 30 min. After incubation, the samples can

be processed by routine protocols for fixation and visualization

of immune-fluorescent stained tissue constructs.

7. Wash three times with PBS+ containing 1% BSA.

8. Fixate with 4% PFA for 5 min at room temperature.

9. Wash three times with PBS.

10. Carefully cut the circular graft open and spread out flat on a

microscope slide.

11. Acquire images or use the section for further staining (e.g.,

Phalloidin). Exemplary images are shown in Fig. 3.

3.9

Phalloidin—F-

Actin Staining

Visualization of the cytoskeleton by widely used f-actin fiber stain-

ing allows for a quick and easy evaluation of morphological char-

acteristics regarding the spreading and orientation of distributed

216

Max Wacker et al.