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International Journal of Bioprinting Expanding 3D cell proliferation with DLP bioprinting

a uniform cell distribution. We observed the internal The microchannel sizes of SMH, MMH, and LMH
cell morphology within the hydrogel and analyzed cell were then analyzed for each sample; the sizes of SMH,
viability (Figure 3B). The observation results over time MMH, and LMH were 249.4 ± 6.86 μm, 487.4 ± 42.59
showed that on day 1, a cell viability of 76.88 ± 6.22% μm, and 748.0 ± 27.86 μm, respectively (Figure 4C).
was achieved. This could be attributed to the loss of The three different sizes of microchannel hydrogels
initial viability caused by cell encapsulation stress, were close to the desired diameter. The observed slight
nutrient restriction, drying, or temporary swelling during differences could be attributed to shrinkage during
printing. The cell viability showed a gradual increase on freeze-drying. The microchannels effectively enhance
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15
days 3, 5, and 7, reaching 79.85 ± 3.51%, 85.72 ± 2.61%, cell viability by providing nutrients within the scaffold
and 87.38 ± 1.04%, respectively, but did not exceed while promoting cell adhesion, growth, and migration, as
90%. However, the cell viability showed significant well as providing space for mass transport. 22-25 Based on
improvement on days 14, 21, and 28, reaching 90.41 ± the results of this study, we predicted that the carefully
9.33%, 95.74 ± 3.26%, and 98.48 ± 0.81%, respectively, fabricated microchannels would provide not only a
which surpassed 90%. A statistically significant increase supply channel for oxygen and nutrients but also a space
in the cell viability was observed on day 35 compared for cell proliferation.
to day 1, with cell viability of 98.89 ± 1.7% (p < 0.001;
Figure 3D). Other reported studies have also shown that 3.5. Analysis of immunostaining in outer
GelMA hydrogel scaffold encapsulating fibroblasts as well microchannel and inner microchannel upon
as various cells such as human hepatocellular carcinoma introduction of microchannels of three different
and human umbilical endothelial cells exhibit good cell sizes in DLP-printed 3D hydrogel scaffold
viability, diffusion, and proliferation for 7 days, indicating The hydrogel scaffolds were DLP-printed with
high biocompatibility of the scaffolds. Printed GelMA is three different sizes of microchannel, and cells were
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known for being highly compatible with cells and did not encapsulated within the hydrogels. The purpose of this
exhibit cytotoxicity. Moreover, the cells encapsulated in experiment was to observe the cell behavior in outer
the F-GelMA hydrogel showed high uniform distribution microchannel (O.M.C) and inner microchannel (I.M.C)
and biocompatibility. This indicates that DLP-printed in each scaffold group. Cell encapsulation was followed
F-GelMA has the potential to serve as a scaffold for cell by 3 days stabilization period. Subsequently, we analyzed
adhesion and proliferation. the α-tubulin/DAPI immunocytochemistry of the O.M.C
and I.M.C of sample cross-sections from 5 to 35 days
3.4. Fabrication and accuracy analysis of three (Figure 5A-(a), (b), and (c)). All size groups successfully
different sizes of microchannel in a DLP-printed 3D encapsulated cells during the O.M.C time course.
hydrogel scaffold However, single-cell morphology was observed, and no
Here, we investigated the viability of cells on a DLP- network formation or cell adhesion and proliferation
printed hydrogel scaffold. While we confirmed that the were observed during long-term culture. No significant
scaffolds exhibit high cell viability, challenges remain as (ns) difference in α-tubulin confluency was observed over
DLP printing is unable to address the microscale barrier the time course of cell encapsulation within the O.M.C
structures that impede cell adhesion and proliferation (ns; Figure 5B, D, and F). This lack of proliferation was
(Figure 2D). Three different sizes of microchannels suitable likely due to the flow and supply restriction of the media,
for increasing cell adhesion and proliferation within the which facilitates nutrient supply to the interior of the
same volume were introduced to facilitate bulk transport encapsulated cells. 64,65
of scaffold cell contents, enhance cell viability, and improve The α-tubulin confluency in the I.M.C showed the
cell proliferation. 23,24,63 The 3D hydrogel scaffolds were following results during the entire time course (5, 7, 14,
printed with small microchannel hydrogel (SMH; 250 μm), 21, 28, and 35 days): 7.11 ± 2.15%, 7.52 ± 1.77%, 8.58 ±
medium microchannel hydrogel (MMH; 500 μm), and 2.16%, 12.32 ± 2.98%, 13.21 ± 2.05%, and 9.22 ± 1.67%
large microchannel hydrogel (LMH; 750 μm).
in SMH, respectively (Figure 5C). A significant difference
To fabricate microchannel hydrogels of three different was observed in SMH between 5 and 28 days (p < 0.05).
sizes using DLP printing technology and to measure In MMH, α-tubulin confluency increased to 4.53 ± 1.08%,
their accuracy, lyophilized samples were prepared for 9.20 ± 3.53%, 12.09 ± 0.83%, 13.72 ± 4.37%, 15.24 ± 4.17%,
SEM analysis (Figure 4A). For the measurement of three and 17.78 ± 0.94% during the time course, and statistically
different sizes of microchannels, the top-view overall significant differences were observed between 5 and 21
image and magnified image of the microchannel were days and 28 and 35 days (p < 0.05, p < 0.01, and p < 0.001;
acquired. In addition, transverse sections were prepared Figure 5E). Similarly, in LMH during the same time course,
to capture images of each microchannel size (Figure 4B). α-tubulin confluency increased to 8.31 ± 1.16%, 8.17 ±

Volume 10 Issue 3 (2024) 416 doi: 10.36922/ijb.2219