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Ng, et al.
range of printable materials and rapid fabrication speed, viability of the printed cells and improves the printing
the drop-on-demand (DOD) material jetting approach outcome by mitigating droplet splashing. Furthermore, it
is attractive for contactless deposition and patterning of is important to limit the printing duration for each printed
different types of living cells and biomaterials within layer within 2 min to prevent excessive droplet evaporation
each layer to achieve improved cell-cell and cell-matrix to maintain high cell viability. The cells were printed using
interactions [30-33] . the recommended printing parameters – cell concentration
Despite the advances in DOD material jetting-based of 4 million cells/mL within printing duration of 2 min; the
bioprinting techniques, there is limited understanding of cells became elongated on day 1 and proliferated well over
how the viability of the printed cells are affected. Droplet a period of 7 days to reach almost 90% cell confluency
impact of cell-laden bio-inks during DOD bioprinting on day 7. The study has highlighted that controlling the
process affects the quality of the printing outcome droplet impact velocity and droplet evaporation is critical
(through droplet splashing) and the cell viability. for achieving improved short-term cell viability and long-
Although the droplet splashing phenomenon has been term cell proliferation of printed cells. The ability to
studied extensively for more than 140 years , in-depth maintain high cell viability and proliferation rate of the
[34]
and high-resolution studies were only conducted in the printed cells is useful for various bioprinting applications,
last 2 decades due to the advancement in the high-speed such as fundamental studies of cell-cell or cell-matrix
video technology . The outcome of the droplet impact interactions, and fabrication of in-vitro tissue models.
[35]
can be categorized into 6 different scenarios – deposition,
prompt splash, corona splash, receding breakup, rebound, 2. Experimental section
and partial rebound. To date, there are only limited studies 2.1. Cell culture
that investigated the influence of droplet impact on the
cell viability of the printed cells [36-39] . Several models Primary human dermal fibroblasts (HDF) were purchased
have been proposed to simulate the cell viability post from CellnTec Advanced Cell Systems and used in
droplet impact, such as the Newtonian model where the this study. The fibroblasts were cultured in CnT-Prime
cells and the droplets are assumed as Newtonian , or Fibroblast Proliferation Medium (CnT-PR-F, 1% serum
[37]
a compound droplet model with both the cells and the medium supplemented with fully defined growth factors
droplets modelled as viscoelastic fluids with different and co-factors) at a temperature of 37°C. The culture
properties, and the ambient fluid modelled as Newtonian medium was changed once every 3 days. The cells were
fluid . Although these models provide some insights routinely passaged in tissue culture flasks (passages 3
[38]
into how the cell viability might be affected by the fluid – 5), and the adherent cells were harvested using CnT
properties of the bio-ink, experimental results have not Accutase cell detachment solution (CnT-Accutase 100)
been collected to verify the accuracy of the models. The at 90% confluency. Different concentrations of cell-
influence of droplet impact velocity on cell viability is a laden bio-inks were prepared; the detached fibroblast
highly-complex phenomenon; an in-depth understanding cells were suspended in 1× phosphate-buffered saline
TM
of the droplet impact velocity on cell viability would be (PBS) solution – HyClone , 0.0067 M without calcium
2+
2+
useful for DOD cell printing applications. (Ca ) and magnesium (Mg ) to get the desired cell
Another important consideration during DOD concentration (0 – 5 million cells/mL). The PBS solution
bioprinting of cell-laden bio-inks is the influence of was selected in this experiment for the following reasons:
droplet evaporation on the viability of the printed cells (i) it is a biocompatible medium to deposit cell-laden
within the encapsulated droplets. This is a critical aspect droplets for fundamental studies of cell-cell and cell-
of DOD bioprinting that has been overlooked, and there matrix interactions (whereby cell encapsulation in
is a poor understanding of how the droplet evaporation hydrogel matrix may not be desirable); and (ii) various
influences the printed cell viability over time. The droplet hydrogels may be printed at low concentrations using the
evaporation mechanism is a highly-complex process, inkjet bioprinting approach; hence, the PBS solution was
which is dependent on various parameters such as the used to serve as a baseline to understand the influence of
evaporation mode of the deposited fluids, the physical different printing conditions on cell viability.
parameters such as temperature and pressure, the property 2.2. Characterization of bio-inks
of the solvent, and the interactions between solvent,
particles, and substrate . The printability of the bio-inks can be evaluated by
[40]
In this study, we demonstrated using a DOD thermal determining the dimensionless Z value – the inverse of the
inkjet bioprinting system that an increase in the cell Ohnesorge number (Oh), which can be defined as the ratio
concentration resulted in slower droplet impact velocity between the Reynolds number and the square root of the
during jetting of sub-nanoliter cell-laden droplets. The Weber number and is independent of the bio-ink velocity.
decrease in the droplet impact velocity leads to higher To investigate the influence of cell concentration on the

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