The WST-1 assay solution has to be prepared in advance of the

experiment. We used 12.5% of standard WST-1 working solution

diluted in cell culture medium containing 10% FCS (see Note 8).

3.6.1

Perform WST-1

Assay (Modified from

Manufacturer’s

Instructions)

1. Take up 3 ml of WST-1 assay solution for each graft inside a

10 ml Luer Lock syringe.

2. Stop the perfusion.

3. Attach the syringe to one of the 0.2 μm infusion filters

upstream to the graft.

4. Set all corresponding 3-way stopcocks to a position so that the

assay solution can only flow inside the examined graft.

5. Carefully fill the lumen of the opened perfusion path with the

assay solution. Take care about the remaining air, so that it will

function as a watershed to push remaining medium out of the

graft, and fill each graft with WST-1 solution until the water-

shed reaches the downstream 3-way stopcock.

6. Now close the upstream and distal 3-way-stopcock.

7. Let the assay incubate as recommended by the manufacturer

and adjust the incubation time to the used cell type for up to

4 h.

8. Perform the WST-1 assay for the positive controls in the well

plate. In order to norm WST-1 proliferation data to a positive

control, the WST-1 assay has to be carried out with the same

volume per seeded cells per surface area ratio as in the examined

tubes. In our case we seeded 175,000 cells per well in a 12-well

plate. The WST-1 assay was therefore carried out with 437 μl.

9. After 4 h remove the WST-1 solution out of the tubes by

opening all 3-way-connectors so that you will be able to push

out the assay solution with air out of the upstream attached

syringe and collect the assay solution in the already attached

downstream sampling syringe.

10. Open all 3-way connectors toward the perfusion system and

restart the perfusion.

11. Measure the assay solution in a 96-well plate in duplicates for

absorption at 440 nm and use 600 nm as reference wave

length.

12. Calculate the WST-1 data for seeding efficiency as shown

below. Exemplary results of seeding efficiency and cell prolifer-

ation are shown in Fig. 2.

Seeding efficiency ¼

Absorbance at 440 nm  Absorbance at 600 nm

ð

Þsamples

Absorbance at 440 nm  Absorbance at 600 nm

ð

Þpositive controls

In Vitro Colonization of Vascular Grafts

215