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International Journal of Bioprinting Bioprinting of PDAC microtissues for drug screening

bioink reached the proper printing state. In this process, (1:100, Invitrogen, USA) and CK19 (1:50, Huabio, China)
cell-laden beads were produced onto a hydrophobic were added to samples and incubated overnight at 4°C.
membrane under the parameters of 0.1 MPa and 1400 ms. Subsequently, secondary antibodies including Alexa Flour
The center-to-center distance between GelMA hydrogel 594 IgG and 488 IgG (1:200, Invitrogen, USA) were used
beads was fixed at 1000 μm by setting the coordinates of the to stain samples for 3 h at room temperature. Finally, DAPI
hydrogel beads in G-code. As a result, an array of GelMA solution (1:1000) was added for cell nucleus staining and
hydrogel beads around 800 μm was generated. Finally, the incubated for 10 min. Samples were then fully washed with
bioprinted cell-laden GelMA hydrogel beads were photo- PBS and observed by fluorescence microscope.
crosslinked by 405 nm blue light (200 mW cm ) for 20 s
-2
before downstream culture. 2.9. Drug treatment
PDAC microtissues were cultured for 1 week before
2.6. Live/dead cell assay drug treatment. Gemcitabine powder (Sigma, USA) was
Live/dead cell analysis was performed to assess cell separately dissolved in three concentrations of stock
viability of microtissues during the culture time at solution (50, 75 and 100 mM/mL). Then, the stock solution
days 1, 4, and 7. Briefly, samples were stained with a was diluted 1000 times in the culture medium and prepared
live/dead viability kit (Beyotime, China) following the into working fluids at different concentrations, including 50,
manufacturer’s instructions. The samples were incubated 75 and 100 μM/mL. PDAC microtissues were cultured in
at 37°C for 25 min, followed by a thorough wash in the working fluids. Non-drug treatment PDAC microtissues
phosphate-buffered saline (PBS; Gibco, USA) and then were observed as control. After drug administration and
observation under a fluorescence microscope (Nikon, incubation for 72 h, the PDAC microtissues were washed
Ti-U, Japan). Cell viability was determined according to with PBS and then subject to cell viability analysis.
the fluorescence area measured by ImageJ. Specifically, the 2.10. Statistical analysis
fluorescent images of Calcein-AM and propidium iodide
(PI) were respectively processed using ImageJ software, Experimental data from at least three independent
and transformed to grayscale for areas quantitation. experiments are expressed in mean ± standard error.
Cell viability was calculated as the ratio of the area of Graphs were plotted using the GraphPad Prism 9 software
Calcein-AM to the sum of area of Calcein-AM and PI. and embellished using Inkscape. We further analyzed the
significant differences of the data using two-way analysis
2.7. Cell morphology analysis of variance (ANOVA), and P < 0.05 was considered
To characterize the cell morphology and spatial distribution statistically significant.
within the GelMA beads, F-actin staining on different 3. Results
PDAC microtissues was conducted at day 1, 4 and 7 of
culture. Briefly, cell samples were washed using PBS, and 3.1. Printing of GelMA hydrogel beads and
then 4% paraformaldehyde (Beyotime, China) was used to assessment of the microstructures
fix cell for 4 h. Afterward, the samples were washed and In this work, we aimed to produce GelMA beads to support
stained with Alexa Fluor 488 phalloidin (1:200, Invitrogen, pancreatic tumor-stroma microtissues, mainly considering
USA) for 2 h at room temperature. Finally, DAPI solution that the spherical structure of beads allows full circulation
(1:1000, Biosharp, China) was added to stain cell nucleus of nutrients and oxygen [25,26] . Hence, GelMA beads at
for 10 min, which was observed using the fluorescence the concentration of 8% (w/v) were generated utilizing a
microscope after PBS washing for three times. pneumatic extrusion printhead. The printhead worked
under pre-set G-code and printed hydrogel beads onto the
2.8. Immunofluorescence staining
substrate in a programmed intermittent pressure-driven
To confirm the interactions between pancreatic cancer cells mode. The intermittent pressure extrudes a low volume of
and normal fibroblasts, cytokeratin 19 (CK19) and smooth GelMA hydrogel in a limited dispensing time, and round-
muscle actin-α (α-SMA) were used to stain BxPC-3 cells shape beads are produced once the printhead is lifted
and NHDFs at days 1 and 7 of culture. Briefly, cell samples up. The bioprinting system is shown in Figure 2A. It has
were gently washed with PBS, and then fixed with 4% been experimentally proven that the bioprinting system is
paraformaldehyde for 4 h at room temperature. After that, well controllable and capable of producing uniform-sized
samples were permeabilized by 0.25% (v/v) Triton X-100 GelMA hydrogel beads (Figure 2B). We can control the
(Beyotime, China) for 30 min on ice, and blocked with size of the formed GelMA hydrogel beads by adjusting the
5% (v/v) bovine serum albumin (BSA; Sigma-Aldrich, dispensing time in the G-code. As shown in Figure 2C, at
USA) solution for 1 h. Primary antibodies against α-SMA a fixed pressure (0.1 MPa), GelMA hydrogel beads with

Volume 9 Issue 3 (2023) 4 https://doi.org/10.18063/ijb.v9i3.676