Page 520 - IJB-10-3

International Journal of Bioprinting Drop-on-demand laser bioprinting

(ii) flow-enabled removal of residual bubbles resulting Although increasing the laser pulse energy yielded
from the collapse of the main bubble; (iii) flexibility in results similar to the initial LIST configuration, 23-26 the
controlling bioink shear rate (scales with the volumetric laser pulse energy threshold for ejection is two times lower,
flow rate); and (iv) prevention of cell agglomeration in cell- and the droplet size is 1.5 times larger. This is likely because
laden inks, a phenomenon observed in long-term printing the bubble is generated much closer to the nozzle (~250
due to gravity in the initial design. μm) compared to the edge of the capillary (~500 μm) in the
initial configuration. One might expect that the conversion
These advancements led to notably enhanced printing of bubble energy to jet kinetic energy is more efficient for
stability, as indicated by the decrease in variation of a shorter bubble–nozzle distance. This effect has been
cell density in printed drops compared to the initial previously observed in works exploiting laser-induced flow
setup. Specifically, when analyzing the normalized cell focusing for jet generation and drug ejection.
27
drop density for both approaches over 1 h of printing, a
significant decrease in the coefficient of variation was 3.3. Impact of ink flow rate on printing workflow and
observed within the redesigned setup in comparison to jet directionality
the initial configuration (41% vs. 151%, p = 0.016) (Figure Using the same model ink, we aimed to establish the
S1 in Supplementary File). Furthermore, using glycerol- maximum flow rate for stable printing—a critical factor
influencing ink viscosity in shear-thinning inks and a
based model inks, we found that the printability range is decisive factor in determining the potential maximum
significantly increased in the redesigned LIST compared printing speed. In our initial experiments, we observed that
to the initial implementation in terms of maximum the ink tended to leak through the nozzle under perfusion
ink viscosity (140 mPa·s vs. 32 mPa·s) (Figure S2 in pressure when flow rates exceeded 300 µL/min. Conversely,
Supplementary File). during withdrawal pressure, occasional air suction through

3.2. Impact of laser pulse energy and ink flow rate the nozzle introduced bubbles into the ink. Consequently,
on droplet size and shape we identified 300 µL/min as the maximum flow rate for
Prior to initiating cell printing, we examined the new LIST this specific capillary geometry and further examined if
flow rates affected jetting directionality.
printing architecture using a model ink consisting of a
water and Allura Red mixture. We investigated the effect Jet angle measurements at 80 μJ laser energy and flow
of key printing settings, such as capillary flow rate, driving rates of 90 and 300 µL/min indicated that the mean change
pressure (i.e., perfusion/withdrawal), and laser energy, on in the jet angle ranged between -1° and 1° (Figure 3).
the printing process. We focused on determining drop These findings suggest that flow rate variation within
size and circularity, which are determinants of printing this range is unlikely to significantly affect bioprinting
resolution. precision and accuracy. A jet angle divergence of 1° results
in a 9-micron divergence in drop deposition, considering
To accomplish this, we printed droplets on microscope the typical capillary-to-sample distance of 0.5 mm. This
slides at a 20 Hz printing speed, employing a range of laser divergence represents a negligible loss of accuracy given
pulse energies (20–120 μJ) and flow rates (6–90 μL/min) the typical drop size (300 µm). Importantly, a flow rate
under perfusion or withdrawal pressure flow conditions of 300 µL/min is 50-fold the liquid ejection volume for
(Figure 2). For flow initiation, pressure > 1 atm was applied drops printed at 20 Hz, suggesting that higher printing
in the perfusion pressure configuration (Figure 2A), speeds may be achievable with a laser operating at
whereas pressure < 1 atm was applied in the withdrawal a higher repetition rate. Finally, the negligible effect
pressure configuration (Figure 2E). We determined that of the flow rate on the jet directionality suggests that
the threshold for achieving stable printing conditions is 40 jetting dynamics are primarily influenced by the bubble
μJ, whereas printing at 100 μJ or higher energy resulted in dynamics for the examined setting.
splashing (Figure 2B and F). The droplet radius increased 3.4. Human umbilical vein endothelial cell
progressively with increasing laser pulse energy (Figure viability is maintained in redesigned
2C and G) (p < 0.001), while droplet circularity exhibited laser-induced side transfer
an inverted relationship with laser energy (Figure 2D and Despite the established viability of primary cell types in
H) (p < 0.001). These findings were consistent in both flow LIST, 23-26 it remained unclear whether the redesigned LIST
configurations. Notably, the printing workflow remained approach would yield similar outcomes. LIST involves cell
stable for a flow rate of up to 90 μL/min, representing exposure to both laser irradiation and thermomechanical
15 times the rate of ink loss via drop ejection at 20 Hz, stress, including (i) long-term (few minutes) shear stress
considering an average drop volume of 5 nL. from bioink flow inside the capillary and loading tube,

Volume 10 Issue 3 (2024) 512 doi: 10.36922/ijb.2832