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Effect of Bioprinting-Associated Shear Stress and Hydrostatic Pressure
brown algae, BioReagent, suitable for immobilization suspension was prepared (alginate concentration 1.5% w/v)
of microorganisms, Sigma-Aldrich, St. Louis, USA) with a cell concentration of 1 million cells/ml (HUVEC).
solutions were prepared in sterile demineralized water or Viscosity measurements were performed on a rotational
sterile PBS (Gibco by Life Technologies, Carlsbad, USA), viscometer (Kinexus ultra+, Malvern Instruments Ltd.,
respectively. The hydrogel solutions were prepared by Malvern, United Kingdom) using a 4° cone geometry. The
autoclaving (agarose) or by stirring overnight (alginate). shear rate was continuously increased according to a defined
Whereas agarose gel was used in combination with range from 0.01 to at least 1,000 s within a period of 3 min
−1
collagen for the 3D cell culture experiments, alginate during which the viscosity and shear stress were measured.
solution was used in the printing procedures due to its
ease of handling and its temperature-independent and 2.5. Droplet volume
thus controllable gelation behavior. The collagen solution The measurement of the droplet volume and all other
was prepared by mixing eight parts of collagen (FibriCol, printing experiments were conducted on a custom-made
Type I Bovine Collagen Solution, 10 mg/ml, Advanced microvalve-based 3D printer for DoD bioprinting of
BioMatrix, San Diego, USA) with two parts medium and hydrogel-cell suspensions, as previously described .
[10]
neutralizing with 1 M sodium hydroxide. PI-FDA solution Droplet measurements were performed as follows. Empty
was used for live-dead staining. For this step, 0.025 g micro test tubes (1.5 ml) were weighed and then loaded with
propidium iodide (PI; 95 % HPLC, Sigma-Aldrich, St. 20 droplets of hydrogel suspension (1 million cells/ml for
Louis, USA) was dissolved in 5 ml Ringer’s solution and alginate 1.5% w/v) at different printing pressures (0.5 and
0.05 g fluorescein diacetate (FDA; Sigma-Aldrich, St. 3.0 bar). The opening time of the microvalve was set to
Louis, USA) was dissolved in 10 ml acetone. 10 µl of 4500 µs and was not changed during the measurement
the prepared PI and FDA solutions were separately mixed (SMLD 300G, Fritz Gyger AG, Gwatt, Switzerland;
with 600 µl Ringer’s solution in micro test tubes. valve diameter 150 µm, schematic view of the valve in
Figure 1C). Subsequently, the test tubes were weighed
2.3. Numerical simulation again, and the average droplet volume was calculated by
Ansys CFX 19.2, a software that uses finite element dividing the difference in weight by the number of dispensed
method to solve the governing equations, was used for droplets and the density of water at 25°C. Due to the low
the numerical simulations. For this purpose, the fluid gel concentrations, it was assumed that the density of the
was considered incompressible, isothermal condition hydrogel solution used was comparable to that of water.
was imposed, and the maximum RMS residual error of
10 was chosen. We set a high-resolution scheme for the 2.6. Post-printing cell viability
−4
advection term discretization and a second order backward An alginate solution with a concentration of 1.5% w/v
Euler scheme for the transient term discretization . The was prepared containing 1 million cells/ml (HUVEC).
[11]
geometry of a mechanical microvalve (SMLD 300G, Fritz This cell-alginate suspension was then transferred to
Gyger AG, Gwatt, Switzerland; valve diameter 150 µm) the cartridge of the 3D printer, which had previously
was considered for the simulation. This valve consists of a been disinfected with ethanol (70% v/v). Then, the cell
stationary piston, a moving piston, and a spring that ensures suspension was dispensed through a magnetic microvalve
microvalve closure. By imposing a high upstream pressure, (SMLD 300G, Fritz Gyger, Gwatt, Switzerland; valve
the cell suspension is placed under pressure and as soon diameter 150 µm) at different pressures (0.25, 0.5, 0.75,
as the valve opens, a drop is ejected. For the numerical 1.0, 1.25, 1.5, 2.0, 2.5, and 3.0 bar) with an opening time
simulation, we used a non-uniform mesh with higher mesh of 4500 µs. At each printing pressure that was set, 20 µl of
density at the entrance of the nozzle and boundary layer the cell suspension was collected in an empty micro test
mesh at the walls. To ensure the use of proper element tube. Subsequently, 7.5 µl was taken from each sample,
size, the grid study was performed on at least three cases pipetted onto a microscope slide, and mixed with an
of fine, medium, and coarse meshes considering the wall equal volume of PI-FDA solution. The samples were then
shear stress as a sensitive parameter. Transient simulation covered with a round cover slip. Imaging was performed
within the valve opening time (4,500 µs) was considered using a fluorescence microscope (Axio Imager M2M, Carl
and the total flow rate during this period (drop volumetric Zeiss, Oberkochen) at five-fold magnification. For each
size) was compared with experimental measurements (see sample, three images were taken at different positions.
below). Two different upstream pressures (0.5 and 3 bar) Non-printed cell-alginate suspension that was taken from
were considered for the simulations. the printing cartridge served as control.

2.4. Viscometry 2.7. Expression of cell-specific markers
Viscosity data were used as input for the simulation of the A cell-alginate suspension containing 1 million cells/ml
alginate flowing inside the microvalve. A cell-alginate (HUVEC) was prepared and dispensed dropwise through
98 International Journal of Bioprinting (2022)–Volume 8, Issue 4