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Protein Nanoparticles Promote Cell Growth in 3D Bioprinted Constructs
tissue constructs [3,4] . The printing of these cellularized by promoting the diffusion of oxygen, gas vesicles were
structures through the precise layering of cell-laden bioinks printed homogeneously throughout the constructs. The
is accomplished using material jetting bioprinting (inkjet, bioprinting experiments were conducted with our novel
microvalve, acoustic, and laser-assisted bioprinting), 3D bioprinting system using our ultra-short peptide
material extrusion bioprinting, and vat polymerization hydrogel [37-41] . The printability of peptide bioinks and
bioprinting (stereolithography, digital light processing, and biocompatibility with various cell types was tested [42,43] .
two-photon polymerization) [5-13] . While each printer has its As gas vesicles have been reported to promote cell
[44]
advantages over the others, there remain many challenges activity in cell culture , this work aims to see if those findings
to face before this technology can be translated into clinical can be extended to attain better outcomes in 3D bioprinting.
practice, regardless of the printer setup . The biocompatibility of the gas vesicles was tested with human
[14]
Two of the most significant issues with 3D bioprinting embryonic kidney cells in both two-dimensional (2D) and 3D
are identifying suitable bioinks and ensuring adequate cell cultures. In addition, the printability of the gas vesicles
nutrient supply to the cells [15-20] . While a great deal of was assessed to ensure that the nanoparticles are capable of
effort has been focused on developing novel biomaterial withstanding the shear stress involved in the printing process.
candidates, it has been much harder to find a solution to The bioink and scaffolding material used throughout this
address the diffusion barrier associated with the printed study was IK self-assembling peptide. Once the printability
6
construct. The diffusion gradient is an inherent result and biocompatibility of the gas vesicles were established,
of the nature of the bioink scaffolding material, which the cell viability and morphology of cells printed with and
hinders the migration of nutrients, ranging from growth without gas vesicles were assessed. The results are promising
factors to oxygen, to the center. This diffusion barrier and suggest that the 3D printing of gas vesicles may positively
must be overcome as delivering these nutrients ensures affect cell activity for up to 7 days when printed together.
the cells’ survival, proliferation, and differentiation.
As such, oxygen-releasing biomaterials have been 2. Materials and methods
explored as a means of promoting its diffusion throughout 2.1. Materials
the construct. Most of the oxygen-releasing biomaterials
developed so far involve scaffolds integrated with peroxides The self-assembling peptide IK (Ac-ILVAGK-NH2)
6
and fluorinated compounds in the form of liquids or solid was custom synthesized by Bachem AG (Budendorf,
micro- and nano-particles [21,22] . Although early reports of Switzerland). Human embryonic kidney cells (HEK293)
these materials are promising, they all require the insertion of were purchased from American Type Culture Collection
materials that do not occur naturally within the human body [23- (ATCC; USA). Cells were cultured in medium Dulbecco’s
25] . This introduces additional uncertainty about the fate of modified Eagle’s medium-high glucose (DMEM-HG;
these materials as they lack the inherent biodegradability Gibco Thermo Fisher Scientific, USA). T175 or T75 cell
of proteins, for example. To this end, we propose an culture flasks and 96- and 48-well plates were purchased
alternate solution to promote oxygen diffusion using gas from Corning, USA. Halobacterium sp. NRC-1 was
vesicle nanoparticles (GVNPs). Gas vesicles are hollow obtained from Carolina Biological Supply (Burlington,
+
gas-filled proteinaceous intracellular organelles common NC, USA) and cultured in CM medium containing 4.3M
to many species of bacteria and archaea . In nature, gas NaCl and trace metals at 42°C with shaking as previously
[26]
[45]
vesicles promote floatation and the availability of oxygen described . H. volcanii H1895 and its corresponding
in the microbial cell . These cylindrical- or spindle-shaped vector pTA963 were kindly provided by Dr. Thorsten
[27]
organelles have canonical ends and vary in size depending Allers (Institute of Genetics, School of Biology, University
on the organism [28,29] . Over the years, GVNPs have drawn of Nottingham, Queen’s Medical Centre, Nottingham,
interest in biotechnological and biomedical applications. This UK). H. volcanii and derivatives were cultured in the
includes traditional nanoparticle applications as drug delivery Hv-YPC medium at 45°C with shaking as previously
systems and other applications based on their unique physical described [46,47] . For solid media, 2% (w/v) agar was
®
properties. One group, in particular, the Shapiro group at added. The CellTiter-Glo luminescent 3D cell viability
®
Caltech, has found that their sound scattering properties and assay kit, LIVE/DEAD Viability/Cytotoxicity kit and
ability to produce harmonic ultrasound signals make GVNPs Actin Cytoskeleton/Focal Adhesion Staining kit were
especially useful as contrast agents and molecular sensors for purchased from Promega, USA, Life Technologies™,
ultrasound and magnetic resonance [30-35] . USA, and Sigma-Aldrich, USA, respectively.
In this study, we developed a new, efficient system 2.2. Engineering and expression of gas vesicles in
for haloarchaeal Haloferax volcanii gas vesicle expression H. volcanii
that utilizes a combination of attributes to facilitate cheaper
and faster GVNP production at yields high enough to be Superfolder Green Fluorescent Protein (sfGFP)
suitable for bioreactor scale [30,36] . To improve cell growth synthetic gene (IDT, Leuven, Belgium) was codon-

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