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Ng, et al.
spreading shear stress. However, we believe the effect of would experience a larger evaporation flux, an increase
viscosity in decreasing droplet impact velocity dominates in the droplet volume from 20 nL to 40 nL (2×) only
in practice; and hence, we observed higher cell viability resulted in a ~1.3× change in diameter from 594.2 ±
when a cell-laden bio-inks of higher cell concentration 4.6 µm to 772.4 ± 5.3 µm. Hence, the 40 nL droplets
are used. would take ~ 50% more time to reach complete dryness
as compared to the 20 nL droplets. All the printed cell-
3.5. Influence of droplet evaporation on printed laden droplets maintained a cell viability of > 92%
cell viability during the first 2 min of droplet evaporation. After
In general, the different variants of droplet evaporation which, there is a significant influence of droplet volume
mode are constant contact radius (CCR) evaporation on the viability of printed cells and the viability of
mode, stick-slide (SS) evaporation mode, or a mixed printed cells decreased significantly from 88.3 ± 2.45%
mode of both CCR and SS evaporation modes [58] . for 40 nL droplets to 48.2 ± 3.54% for 20 nL droplets at
The pinning (CCR evaporation mode) and depinning 4-min interval (Figure 7A). Low cell viability of <50%
(SS evaporation mode) of the droplet’s contact line was observed for all cell-laden droplets (20 – 40 nL) at
depends on the Young’s unbalance force. A low 6-min interval and beyond (Figure 7B). The constant
Young’s unbalanced force leads to CCR evaporation droplet evaporation led to a more hypertonic, i.e., “high
mode, whereas a high Young’s unbalanced force leads salt” environment and thus resulted in higher cell
to SS evaporation mode. In the SS evaporation mode, apoptosis over time [62] . Hence, it is important to strike
the contact line remains pinned for a period and then a balance between achieving high printing resolution
slides to form a smaller radius repeatedly. The sliding of and maintaining high cell viability. A higher printing
contact line is triggered when the unbalanced Young’s resolution (smaller droplet volume) would lead to lower
force is too high, and a new equilibrium is reached with cell viability due to the droplet evaporation process
a smaller drop radius due to the less deviation from that leads to an unfavorable hypertonic environment
the equilibrium contact angle [59] . The contact angle of for the encapsulated cells. Hence, it is recommended to
the liquid drop reduces as the evaporation progress, deposit a minimum droplet volume of 20 nL and limit
resulting in the increase in the unbalanced Young’s the printing time of cell-laden droplets for each printed
force. CCR evaporation mode experiences a constant layer within 2 min for 20 – 30 nL droplets and within
evaporation flux over time, whereas SS evaporation 4 min for 40 nL droplets to achieve a high cell viability
mode experiences decreasing evaporation flux over of > 85%.
time [60] . Hence, droplets of the same volume would 3.6. Long-term printed cell proliferation study
evaporate faster under CCR evaporation mode as
compared to SS evaporation mode. Two critical steps (droplet impact velocity and
Although different hydrogels may be printed droplet volume) within the DOD bioprinting process
together with the cells in the thermal inkjet print-head, have been identified in this study that play important
the printable polymer concentration is typically low at role in influencing the viability of the printed cells.
1% w/v or lower. Hence, the PBS solution used in this A cell-laden bio-ink with higher cell concentration
study serves as a baseline to understand the influence of (4 million cells/mL) leads to an overall slower droplet
droplet evaporation on cell viability. It was observed that impact velocity (5.77 m/s); this helps to mitigate
the cell-laden droplets experienced CCR evaporation the degree of droplet impact-induced damage to the
mode as the contact line remained pinned during the encapsulated cells. Next, the droplet evaporation
evaporation of the cell-laden droplets. As a result, the study in this work has shown that droplet evaporation
evaporation flux remained constant throughout the over time leads to an unfavorable hypertonic
evaporation mode as there is no change in the droplet environment which causes potential cell death that is
diameter. The evaporation flux for CCR evaporation apoptosis process (Figure 8A). Hence, the following
mode in this study can be expressed as : parameters were selected to evaluate the long-term cell
[61]
proliferation profile of the printed cells: a cell-laden
Ù
− () =mt 4 D(1 − H cR) v (7) bio-ink with a concentration of 4 million cells/mL
(to achieve the lowest droplet impact velocity) and a
where D is the diffusivity of the vapor in the air, printing duration of <2 min (to mitigate the dehydration
H is the relative humidity of the ambient air, c is the of printed cells). The cell-laden droplets were printed
v
vapor concentration and R is the contact-line radius. as 8 × 8 array of droplets (30 nL droplet volume per
The 10 nL droplets evaporate rapidly within 2 min, spot) and cultured over a period of 7 days to evaluate
hence only larger droplets are used for the droplet its proliferation profile. There is no negative control
evaporation study. Although a larger droplet volume (non-printed cells) for this study as the manual hand-

International Journal of Bioprinting (2022)–Volume 8, Issue 1 35