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Effect of Bioprinting-Associated Shear Stress and Hydrostatic Pressure
1:100 in 3% bovine serum albumin (BSA) solution and previously described for the 2D experiments (HUVEC).
50 µl of the primary antibody solution was pipetted For each printing parameter setting (0.5 and 3.0 bar),
into each well (one primary antibody per well). The cell a separate micro test tube was filled with 1.6 ml cell-
culture plates were incubated for 1 h and then washed alginate suspension. After printing, the suspension
with PBS at room temperature (3 times for 15 min). was centrifuged and the supernatant removed. Each
Accordingly, the secondary antibodies were diluted 1:100 cell pellet was suspended in 50 µl cell culture media
in a 3% BSA solution and added to the wells. The plates leading to a final cell concentration of 32 million cells/
were incubated and washed again. Finally, 50 µl of a ml. Meanwhile, an agarose, a collagen solution, and a
DAPI solution (diluted 1:10,000 in PBS) was added to hMSC-medium suspension with a cell concentration of
each well. After an incubation period of 2 min at room 32 million cells/ml were prepared. Agarose, collagen,
temperature, the samples were washed with fresh PBS and both cell suspensions were mixed in such way to
again (three times for 15 min). Imaging was conducted achieve a final cell concentration of 3 million cells/ml
on a fluorescence microscope (Axio Imager M2M, Carl (per each cell type), an agarose concentration of 0.2%,
Zeiss, Oberkochen). The primary antibodies used were and a collagen concentration of 0.5 % (Agr0.2Coll0.5).
CD144 Monoclonal Antibody and VWF Monoclonal After 14 days of incubation at 37°C and 5% v/v CO ,
2
Antibody (both ThermoFisher Scientific, Waltham, USA). HUVECs were immunofluorescently labeled as follows.
The secondary antibodies used were Goat Anti-Mouse IgG The cell-laden hydrogel samples were fixed in ice-cold
H&L Alexa Fluor 594 (ThermoFisher Scientific, Waltham, methanol (VWR International, Leuven, Belgium) for
USA) with an optimum excitation wavelength at 590 nm 15 – 20 min at room temperature. The primary antibody
and an emission maximum at 617 nm, and Goat Anti-Rabbit (Monoclonal Anti-CD31 PECAM-1, Sigma-Aldrich, St.
IgG H&L Alexa Fluor 488 (ThermoFisher Scientific, Louis, USA) was diluted with a BSA solution (3% in
Waltham, USA) with an optimum excitation wavelength at PBS) to a ratio of 1:100. After removal of the methanol,
495 nm and an emission maximum at 519 nm. the samples were washed in PBS for 5 min and rinsed in
300 µl of the primary antibody solution for 48 h at 37°C.
2.8. Pre-vascularization potential of HUVEC After removing the supernatant, the samples were washed
twice in PBS at 37°C to remove excess antibody (first for
The pre-vascularization potential of printed endothelial 5 min, then for 12 – 24 h). The PBS was removed and the
cells was evaluated in 2D as well as in 3D cell culture secondary antibody (Goat Anti-Mouse IgG H&L Alexa
experiments. In the 2D study, a basal membrane matrix
(Geltrex™ LDEV-Free Reduced Growth Factor Basement Fluor 594, Thermo Fisher Scientific, Waltham, USA) was
Membrane Matrix, ThermoFisher Scientific, Waltham, diluted in BSA solution (1:400) and added to the wells,
which were then incubated for 48 h at 37°C. The samples
USA) was slowly thawed at 4°C overnight. Then, 90 µl were subsequently washed with PBS three times. DAPI
of Geltrex™ was pipetted into a 48-well plate that was staining was conducted as described above. Finally, the
incubated for 30 min at 37°C to induce gelation of the samples were washed with PBS again 3 times (5 min each
matrix substrate. A cell-alginate suspension was prepared time) and stored in fresh PBS at 4°C until imaging by
(1.5% alginate, 1 million cells/ml, HUVEC), transferred two-photon laser scanning microscopy (TPLSM).
to the 3D printer, and printed into centrifugation tubes at
different static pressures (0.5 and 3.0 bar) with a constant 2.9. Statistical analysis
valve opening time of 4500 µs (SMLD 300G, Fritz Gyger
AG, Gwatt, Switzerland; valve diameter 150 µm). Then, Statistical analysis was performed using a one-way
500 µl of each printed suspension was added to micro test analysis of variance with Tukey’s post hoc test using
tubes previously filled with 4,500 µl cell culture media. SPSS software (Version 25, IBM Corporation, Armonk,
After mixing, 500 µl of each diluted cell suspension was USA). A value of P < 0.05 (*) was considered statistically
separately pipetted into the prepared wells and the cell significant and a value of P < 0.005 (**) was considered
culture plates were incubated at 37°C and 5% v/v CO . highly statistically significant. The number of replicates
2
Live-dead imaging of the samples was conducted on the is given in the respective figure caption.
2 day of incubation (Observer Z1, Zeiss, Oberkochen, 3. Results
nd
Germany). The images of the PI-FDA-stained cells were
analyzed by counting the number of crossings, closed The viscosity curves of the cell-laden alginate solution
networks, and extensions formed by the cells using applied in this study clearly confirmed the shear thinning
ImageJ and the Angiogenesis Analyzer plug-in freeware behavior typical for this hydrogel (data not shown). The
tool developed by Gilles Carpentier. viscosity continuously decreased with increasing shear
To investigate the pre-vascularization potential of rate from 0.01 to 1000s . Based on these experimentally
−1
printed cells in a 3D environment, cell-alginate suspension derived viscosity curves, the flow behavior index n and
was prepared and dispensed through the microvalve as the consistency index K used to describe the rheological

100 International Journal of Bioprinting (2022)–Volume 8, Issue 4