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Controlling Droplet Impact Velocity and Droplet Volume Improves Cell Viability in Droplet-Based Bioprinting
properties (Z values) of the cell-laden bio-inks, cell-laden the nozzle-substrate distance of ~ 15 mm. The average
bio-inks of varying cell concentrations (0 – 5 million droplet velocity profile can be obtained by calculating
cells/mL) were prepared. Measurements were performed the distance travelled by the droplets (n = 15) between
on the different cell-laden bio-inks to investigate the subsequent frames (10 µs apart). Furthermore, the camera
influence of cell concentration on viscosity, surface is also focused on the substrate surface to capture high-
tension, and density of the bio-inks and their respective speed images of the droplet impact on substrate surface
Z values. The rheological properties of the cell-laden at varying cell concentration (0 – 4 million cells per mL)
bio-inks were evaluated using the Discovery hybrid using 144,000 fps, 5× zoom and 1/950,000 shutter speed.
rheometer (TA instruments, New Castle, DE, USA). The
values of the strain amplitude were first verified to ensure 2.5. Influence of droplet impact on printed cell
that all measurements were performed within the linear viability
viscoelastic region. Next, the viscosities of different cell-
laden bio-inks were evaluated for shear rates ranging The thermal inkjet printer (HP D300e Digital
from 10 to 10 s at a constant temperature of 25°C. Dispenser) was utilized to dispense cell-laden droplets
4
−1
2
The surface tension of the bio-inks was measured using (1 – 4 million cells/mL) directly onto dry tissue-treated
Optical contact angle system (OCA 15 EC, Data Physics culture plate across a nozzle-substrate distance of ~
Instrument), and a weighing balance was used to measure 15 mm to investigate the influence of droplet impact
the density of the bio-inks (weight per mL of bio-ink). on printed cell viability at varying cell concentrations.
A sample size of 5 was used for all the measurements. The cell-laden droplets were printed at 1 kHz frequency
into tissue-treated 12-well plates to obtain 8 × 8 array
2.3. Evaluation of bio-inks of droplets (20 nL per spot) in each of the 12-well plate
(both dry well-plates – original well-plates and the filled
A thermal inkjet printer (HP D300e Digital Dispenser) well-plates – original well-plates filled close to the brim
was utilized for cell printing; cell-printing cassettes with PBS solution). The total printing time for each cell-
(specially-designed C-8 cassettes with 8 embedded laden bio-inks (1 – 4 million cells/mL) is <2 min per
thermal inkjet print-heads with nozzle orifice of 80 µm well plate. The printed arrays of cell-laden droplets were
diameter were used in this study) with a high printing immediately evaluated for its cell viability by adding
frequency of 1 kHz were used in this study. The thermal Live/Dead Viability/Cytotoxicity kits (Invitrogen™
inkjet print-head dispensed a constant droplet volume
of ~0.345 nL, and multiple droplets were printed at the L3224, Thermo Fisher Scientific) directly and incubating
same spot to achieve the desired droplet volume. The for 10 min before fluorescence imaging. The stained green
nozzle to substrate distance is approximately 15 mm. cells represent viable printed cells, whereas the stained red
cells represent dead printed cells. The average printed cell
Different cell-laden bio-inks (1 – 5 million cells/mL)
were printed directly onto dry tissue-treated 12 well viability (%) is obtained by calculating the ratio of viable
plates at varying total dispensed volume of 20 nL, 40 nL, green cells to dead red cells inside each printed droplet.
and 60 nL to evaluate its printability and printed cell The droplet impact velocity of each cell-laden bio-inks (1
output. Furthermore, the cell-laden bio-inks (1 – 5 million – 4 million cells/mL) is then obtained from the high-speed
cells/mL) were also printed directly into tissue-treated images in earlier study to analyze the influence of droplet
12-well plates that were filled close to the brim with 1× impact velocity on the viability of printed cells.
PBS solution to analyze the influence of thermal inkjet 2.6. Influence of droplet evaporation on printed
printing process on the viability of printed cells at varying
cell concentrations. cell viability
The thermal inkjet printer (HP D300e Digital Dispenser)
2.4. High-speed imaging of droplet dispensing was utilized to dispense varying volume of cell-laden
A high-speed camera (Photron Nova S12 – up to 200,000 droplets (4 million cells/ml at 20, 30, and 40 nL per
frames per second [fps]) was used to capture high-speed droplet position – the volume of each dispensed droplet
images of cell-laden droplets travelling from the nozzle is ~0.345 nL) directly onto dry tissue-treated culture
orifice until it hits the substrate surface (~ 15 mm apart). plate to investigate the influence of droplet evaporation
As the number of recorded fps increases, it would lead on printed cell viability. One of the key advantages of the
to a smaller area of interest being captured. Hence, the inkjet bioprinting system is its high printing resolution
number of recorded fps is selected based on the highest through deposition of nanoliter droplets. However, there
possible fps for the area of interest. To evaluate the droplet is limited studies that investigate the influence of droplet
velocity profile, the high-speed images were captured at evaporation on printed cell viability in nanoliter droplets.
100,000 fps, 1× zoom and 1/950,000 shutter speed to The understanding of this phenomenon would help to
obtain the full profile of the droplets travelling along implement a suitable printing duration for deposition of

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