Page 84 - IJB-8-3

Protein Nanoparticles Promote Cell Growth in 3D Bioprinted Constructs
and quantified cell viability data are included. In samples growth of the cell aggregates is tracked over 7 days and
analyzed 24 h after bioprinting, about 85.8 ± 4.8% visible in Figure 7A. The growing size of the cellular
of cells were observed to be alive in the control group aggregates over time is evidence for cell proliferation.
without GVNPs, while an increase in viability to 89.7 ± These aggregates increase in diameter, as shown in
3.7 was found in samples with GVNPs at this time point. Figure 5. This finding is consistent with the previous
A significant increase was found in cell viability when study; after 7 days of culture, HEK 293 become dense
measured 4 and 7 days after printing with GVNPs from in structure with multiple cells aggregate up to 140 μm
83.4 ± 4.6 to 92.6 ± 3.7 on day 4 and 91.3± 3.9 to 96.4± in width . Moreover, electron microscopy imaging was
[63]
2.1 on day 7 with P = 0.0005 and 0.009, respectively. performed to visualize the GVNPs in the gel construct
The imaging results in Figure 6A show that after printing. These images depicting the GVNPs in the
the GVNPs did not affect the morphology of the environment surrounding the cells 2 and 8 days after
HEK293 cells. An earlier study reported that the addition printing are shown in Figure 7B. This is in contrast with
of GVNPs to cells in 2D culture was associated with an the lack of gas vesicles present in the imaged control
increase in cell activity . Our results (Figure 6A and B) samples.
[44]
suggest that the cells printed with GVNPs showed better
As we aimed to test the potential for these protein-
viability than the cells printed alone. As the GVNPs based nanostructures in promoting the growth of printed
constitute the thin protein-membrane and the gases inside
and are permeable to gases in the environment, the effect cells, it was crucial to preserve cell viability, morphology,
[64-67]
is likely due to the gas permeability of the protein shell. and function after printing . Therefore, we optimized
One way through which this can occur is by promoting the the 3D bioprinting parameters for HEK 293 cells and
availability of oxygen to the cells as the oxygen is allowed incorporated the GVNPs into the 3D construct by adjusting
to diffuse from within the gas vesicles to the surrounding syringe pump flow rates, inner diameters of the nozzle,
environment. This increased oxygen availability may printing speed, and layer height to mitigate such effect.
render the environment more similar to favorable in vivo The notion that cell growth and death are very strongly
conditions, thereby promoting cell survival. Another affected by the extracellular environment has been well
possibility could be that nearby GVNPs enable cells to documented in the literature . The ECM is crucial to tissue
[68]
clear waste or other species within the environment more engineering, as it dictates cellular morphology and guides
rapidly. Byproducts of cellular respiration include CO , the connections between cells and the site of interactions.
2
so the permeability of the GVNP shell coupled with the Together, the ECM architecture and its composition affect
concentration gradient of the different gases in the system cell growth, connection, differentiation, and adhesion. Our
may have promoted the diffusion of CO away from the results indicate that the GVNPs were able to incorporate
2
cells. within the peptide fibers. The gas vesicles were also
able to stay within the extracellular environment for at
3.6. Morphological study of bioprinted cells least 8 days, possibly increasing the porosity of the gel
Cell aggregates formed within gels after printing, and constructs, thereby allowing more medium and nutrients
their actin cytoskeletons and nuclei were stained. The to penetrate the center.
A B

C

Figure 6. (A) Live/dead staining images for the printed cells with and without GVNPs at days 1, 4, and 7 after printing scale bar 100 μm.
(B) The quantitative analysis of cell viability by ImageJ for the 6 different images. (C) Morphology and 3D distribution of the cells within
printed constructs after 7 days. *P < 0.05, ***P < 0.001.

76 International Journal of Bioprinting (2022)–Volume 8, Issue 3