Air-loaded Gas Vesicle Nanoparticles Promote Cell Growth in Three-dimensional Bioprinted Tissue Constructs

Abstract

Three-dimensional (3D) bioprinting has emerged as a promising method for the engineering of tissues and organs. Still, it faces challenges in its widespread use due to issues with the development of bioink materials and the nutrient diffusion barrier inherent to these scaffold materials. Herein, we introduce a method to promote oxygen diffusion throughout the printed constructs using genetically encoded gas vesicles derived from haloarchaea. These hollow nanostructures are composed of a protein shell that allows gases to permeate freely while excluding the water flow. After printing cells with gas vesicles of various concentrations, the cells were observed to have increased activity and proliferation. These results suggest that air-filled gas vesicles can help overcome the diffusion barrier throughout the 3D bioprinted constructs by increasing oxygen availability to cells within the center of the construct. The biodegradable nature of the gas vesicle proteins combined with our promising results encourage their potential use as oxygen-promoting materials in biological samples.

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization

“…Previous studies have suggested that the GvpC protein is sufficiently flexible in tolerating exogenous sequence inserts [ 7 , 16 , 20 ]. We inserted the Haloarchaeal codon-optimized gene of Superfolder Green Fluorescent Protein (sfGFP) into a loop of the acidic tail of the gvpC gene (Additional file 1 : Figure S2) and expressed it with the GVNP operon in Haloferax volcanii [ 15 ]. sfGFP_GVNPs expressing H. volcanii cells appear pink and opaque on the plate compared to wild-type red transparent colonies (Additional file 1 : Figure S3).…”

“…Halobacterium sp. NRC-1 was cultured in a CM + medium containing 4.3 M NaCl and trace metals at 42 °C with shaking, as previously described [ 7 , 15 – 17 , 36 ]. The GVNP operon from Halobacterium sp.…”

“…For solid media, 2% (w/v) agar was added. The processes for producing and culturing GVNPs were performed as previously described [ 7 , 15 , 17 , 45 ]. H. volcanii lawns or floating cells were grown for 2 weeks and lysed osmotically with PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, and 2 mM potassium phosphate monobasic, pH 7.4) containing 10 mM MgSO 4 and 50 μg/mL of DNase I (Sigma-Aldrich, USA).…”

“…Various aquatic prokaryotes naturally produce GVNPs, enabling them to float in light- and oxygen-rich upper water layers by increasing the microorganism's surface area-to-volume ratio in response to environmental cues [ 2 , 3 , 6 , 10 , 11 ]. Furthermore, over the past decades, GVNPs have been used in various applications, such as biocatalysis [ 7 ], endotoxemia treatment [ 12 ], contrast-enhanced ultrasound [ 13 ], hyperpolarized xenon magnetic resonance imaging [ 14 ], 3D printing [ 15 ], and as biocompatible vaccine vectors in vaccine research [ 9 , 12 , 16 – 18 ].…”

“…NRC-1 is valuable for various processes, although one significant challenge is the slow growth rate, expensive antibiotics, and inconsistent induction systems of the Halobacterium expression host [ 6 , 15 , 21 , 22 ]. Therefore, we recently developed a new, efficient haloarchaeal GVNP expression system using a Haloferax volcanii as a host , with significantly short growth times that enable the production of haloarchaeal GVNP fusions on a larger scale without the need for antibiotics [ 15 ]. Moreover, H. volc anii has well-developed microbiological and molecular genetics techniques [ 23 , 24 ], including a facile DNA transformation system, shuttle plasmids, and selectable markers [ 25 , 26 ].…”