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

Figure 4. Fabrication and accuracy analysis of three different sizes of microchannels in a digital light processing (DLP)-printed 3D hydrogel scaffold.
(A) Three different sizes of microchannel hydrogels made with DLP printing technology (small microchannel hydrogel [SMH], medium microchannel
hydrogel [MMH], and large microchannel hydrogel [LMH]). Scale bars: 1 mm. (B) Scanning electron microscopic (SEM) images of three different sizes of
microchannel hydrogels. The top images were acquired at 30× magnification; scale bars: 1 mm. The middle images were acquired at 100× magnification;
scale bars: 500 µm. The bottom row shows transverse cross-section images of microchannel hydrogels with three different sizes at 100× magnification. Scale
bars: 500 µm. (C) Inner diameter analysis of three different sizes of microchannel hydrogel. Data are shown as means ± SD (n = 5).

1.96%, 12.82 ± 3.96%, 14.04 ± 4.51%, 15.75 ± 1.42%, and function of cells and facilitate the supply of oxygen and
21.01 ± 3.18%, and a statistically significant difference was nutrients to the center of the scaffold, as compared to
observed between 5 and 35 days (p < 0.001; Figure 5G). a non-shaking culture. 66-68 Encapsulation in the O.M.C
resulted in cells maintaining a round morphology without
In the I.M.C, we observed an increasing trend in
α-tubulin confluency from SMH to MMH to LMH. As proliferation, regardless of the media flow environment.
However, in the I.M.C, the media flow environment
reported by others, the presence of microchannels can allowed for the smooth transport of oxygen and nutrients
lead to higher cell viability, increased urea synthesis, and inside the microchannel, leading to high cell proliferation
albumin secretion in adjacent regions of the hydrogel. and network formation (Figure 6A). Throughout the time
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The presence of microchannels also reportedly offers course, α-tubulin confluency in O.M.C remained low at
biocompatibility benefits, such as improved cell penetration 5–10% among different sizes of hydrogels during the long-
throughout the hydrogel scaffold, uniform cell distribution, term culture lasting for 5 to 35 days (ns; Figure 6B, D, and F),
and higher oxygen saturation. Despite these benefits, we consistent with previous experiments. Conversely, cells in
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did not observe a significant increase in cell proliferation the I.M.C formed a network and changed shape during
after the inclusion of microchannels. Even after a long- the long-term culture period. The α-tubulin confluency
term culture period, the α-tubulin confluency did not increased from 11.77 ± 4.07% to 31.91 ± 6.19% from 5 to
exceed 30% for all sizes of O.M.C and I.M.C. Our results 35 days, with statistically significant differences observed
suggest that another effective method may be necessary to between 5 and 35 days (p < 0.05; Figure 6C).
induce cell proliferation.
During the time course of MMH, α-tubulin confluency
3.6. Analysis of immunostaining and cell increased to 21.47 ± 4.65%, 23.36 ± 3.65%, 25.49 ± 1.70%,
proliferation in O.M.C and I.M.C from three different 32.88 ± 14.84%, 39.84 ± 2.51%, and 40.74 ± 2.87%,
sizes of DLP-printed 3D hydrogel scaffolds with a respectively, with a statistically significant difference
change in media flow environment between 5 and 35 days (p < 0.05; Figure 6E). Similarly, at
To facilitate effective cell culture and improve the oxygen LMH, α-tubulin confluency increased to 16.38 ± 2.28%,
and nutrient supply to cells within the microchannels, we 27.13 ± 3.69%, 30.61 ± 0.73%, 36.61 ± 4.03%, 47.84 ± 8.22%,
modified the media flow environment. It has been reported and 71.97 ± 3.32%, respectively, with a statistically significant
that media flow environment could enhance biological difference between 5 and 35 days (p < 0.001; Figure 6G).

Volume 10 Issue 3 (2024) 417 doi: 10.36922/ijb.2219