Page 488 - IJB-10-1

International Journal of Bioprinting Endothelial monolayer formation on scaffolds

2.3. Scaffold coating with fibrin described 19,20 to enable cell alignment with the flow. A
Coating was performed by application of human fibrinogen schematic of the conditions in the slide is depicted in
(10 mg ml in PBS; Sigma-Aldrich, St. Louis, USA) Figure S2 (Supplementary File). After cultivation, cells
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solution for 10 s to the scaffold before crosslinking using were fixed, and fluorescence staining was performed.
5 µl of human thrombin (diluted to 5 U ml in PBS; Sigma- Therefore, scaffolds were incubated with 0.1% Triton-X
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Aldrich, St. Louis, USA) to each scaffold. Crosslinked, (Sigma-Aldrich, USA) for 1 h. Next, the scaffolds were
coated scaffolds were immersed repeatedly in fibrinogen stained with phalloidin (Phalloidin-iFluor 555, Abcam
TM
solution for 10 s followed by 1 h of incubation at room PLC, UK) for 90 min and washed again six times with PBS.
temperature. The initial amount of thrombin was sufficient Incubation with 300 nM DAPI solution (Sigma-Aldrich,
to accomplish the crosslinking after further coating steps. USA) was carried out for 30 min. Subsequently, VE-
cadherin staining was performed by overnight incubation
2.4. Characterization of the fibrin layer with an anti-VE cadherin antibody (rabbit origin, Abcam
The fibrin layer, resulting from repeated scaffold coating, plc., UK). Antibody staining was performed by incubation
was visualized by immunostaining. Therefore, scaffolds for 2 h with conjugated antibody to the first antibody (goat
were fixed with 4 wt% paraformaldehyde for 30 min and anti-rabbit Alexa Fluor® 488; Abcam PLC, Cambridge, UK).
then washed three times with PBS. Scaffolds were incubated Scaffolds were washed thrice with PBS before microscopy.
with an anti-fibrinogen antibody (anti-fibrinogen alpha KLF2 staining was carried out by overnight incubation at
chain antibody; Abcam PLC, Cambridge, UK) overnight 8°C in PBS containing an anti-KLF2 antibody in a dilution
at 4°C before incubation with a fluorophore-linked anti- of 1: 150 (mouse origin; Abcam PLC, UK) after fixation
fibrinogen antibody (Goat Anti-Rabbit IgG H&L Alexa and incubation with Triton-X. Antibody staining was
Fluor® 555, Abcam PLC, Cambridge, UK) for 1 h. Stained visualized by incubation for 2 h with a label-conjugated
scaffolds were evaluated microscopically to evaluate second antibody (goat anti-mouse Alexa Fluor® 555,
fibrin layer thickness and pore size. Image acquisition Abcam PLC, Cambridge, UK).
and analysis were performed using an Olympus IX83
fluorescence microscope in combination with the software Images of cells and scaffolds were captured using
cellSens dimensions (Olympus K.K., Shinjuku, Japan). an Olympus IX-83 inverted microscope. Multichannel
Z-stacking was used to increase the depth of field. fluorescence images were taken with Z-stacking
algorithms to expand the depth of field. In the case of KLF2
2.5. Static and dynamic cultivation of HUVECs on immunostaining, image acquisition was carried out with
MEW and MES scaffolds the same exposure time, gain, and contrast for all images
HUVECs (PromoCell GmbH, Heidelberg, Germany) to obtain comparable results.
were used in all static and dynamic experiments. HUVEC
suspension with a density of 250,000 cells cm in 20 µl The axis rotation and the roundness of the HUVECs
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of EGM2 was added to the scaffolds, which were then were analyzed based on the cell shape visualized through
incubated for 1 h to promote cell adhesion before surplus the VE-cadherin staining. In detail, axis rotation and
cell culture medium was added. Cultivations were roundness were calculated after image processing using
performed at 37°C and 5% pCO . EGM2 (PromoCell Fiji as described in Figures S3 and S4 (Supplementary
2
GmbH, Heidelberg, Germany), supplemented with File). By performing contrast enhancements, threshold
additional 8% fetal bovine serum (Sigma-Aldrich, USA) adjustments, and by using filters such as “find edges” and
and 0.5% gentamicin (10 mg ml , Sigma-Aldrich, St. “unsharp mask,” a binary mask for recognition of structural
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Louis, USA), was used for all experiments. The medium features was created. Ten pictures were analyzed out of
was changed every 2 days during the static cultivation three separate scaffolds from separate cultivations with cell
experiments. numbers ranging from 1265 to 2812 cells per experimental
setup (median = 1856 cells), depending on the conditions
Dynamic cell culture experiments on scaffolds were of the cultivation.
performed using coated µ-slides I Luer 3D (ibidi GmbH,
Gräfelfing, Germany). Scaffolds were cultivated for 7 For the assessment of altered cell roundness under the
days under static conditions and transferred into the influence of flow, the cell area (A) and the longest axis (L)
molds of the µ-slide. The slides were connected to the were measured. Cell elongation was evaluated using the
perfusion system (ibidi GmbH, Gräfelfing, Germany), parameter “roundness” (0 = straight line, 1 = perfect circle)
21
and a steadily increasing laminar flow was applied that according to Brown using the formula:
resulted in a final shear stress of 5 or 10 dyn cm² depending 4 A (I)
on the experiment. Dynamic cultivation was carried out Roundness = L 2
for 3 days in adherence with the protocols as previously

Volume 10 Issue 1 (2024) 480 https://doi.org/10.36922/ijb.1111