Three-Dimensional Bioprinting of Organoid-Based Scaffolds (OBST) for Long-Term Nanoparticle Toxicology Investigation

Abstract

The toxicity of nanoparticles absorbed through contact or inhalation is one of the major concerns for public health. It is mandatory to continually evaluate the toxicity of nanomaterials. In vitro nanotoxicological studies are conventionally limited by the two dimensions. Although 3D bioprinting has been recently adopted for three-dimensional culture in the context of drug release and tissue regeneration, little is known regarding its use for nanotoxicology investigation. Therefore, aiming to simulate the exposure of lung cells to nanoparticles, we developed organoid-based scaffolds for long-term studies in immortalized cell lines. We printed the viscous cell-laden material via a customized 3D bioprinter and subsequently exposed the scaffold to either 40 nm latex-fluorescent or 11-14 nm silver nanoparticles. The number of cells significantly increased on the 14th day in the 3D environment, from 5 × 10⁵ to 1.27 × 10⁶, showing a 91% lipid peroxidation reduction over time and minimal cell death observed throughout 21 days. Administered fluorescent nanoparticles can diffuse throughout the 3D-printed scaffolds while this was not the case for the unprinted ones. A significant increment in cell viability from 3D vs. 2D cultures exposed to silver nanoparticles has been demonstrated. This shows toxicology responses that recapitulate in vivo experiments, such as inhaled silver nanoparticles. The results open a new perspective in 3D protocols for nanotoxicology investigation supporting 3Rs.

Keywords: 3D bioprinter; Calu-3; long-term culture; nanoparticles; nanotoxicology.

Figure 1

Three-dimensional bioprinting process parameters. (A) Left: hydrogel viscosity calculated at different sodium alginate (SA) concentrations for different extrusion temperatures (25–37 °C). Right: extrusion force measured for the different SA at 35 °C. (B) Preserved geometry at different SA: from left to right: 1, 2, 3, 4 and 5%. Scale Bar: 10 mm. (C) Left: minimal extrusion speed threshold able to preserve the geometry in (B) at different SA concentrations. Right: hydrogel viscosity calculated at the minimal extrusion speed for the different SA concentrations at 35 °C.

Figure 2

Study overview and experimental protocol. Experimental protocol for cell-laden multilayer generation, imaging and toxicological assay. Feedback was adopted to better define the 3D printing process parameters. NPs: carboxyl fluorescent nanosphere or polydispersity colloidal silver nanoparticles, 2P: two-photon microscopy. Modified from www.BioRender.com.

Figure 3

Cellular viability in the 3D-printed cell-laden multilayer. (A) Brightfield (left) and calcein-AM-loaded (right) cells at day 1 and day 21, respectively. (B) Cell viability measured at day 1, 7, 14 and 21 in 3D culture. N = 8 for each time-point. Significance set as ** p < 0.01, *** p < 0.001 for live cells (green line) and # p < 0.05 for death cells (red line) vs. control at time 0 (Day-0). Data are expressed as mean ± SD. Scale bars: 1 mm.

Figure 4

Thiobarbituric acid reactive substance assays (TBARS). TBARS for printed Calu-3 cells after hydrogel solubilization after 1 h (T0), and 1, 7 and 14 days in culture. *** p < 0.001 vs. T0. Data are expressed in %mean ± SD vs. T0, represented as percentage vs. control. N = 4.

Figure 5

Cell cycle in unprinted and printed cells. (A) Representative cell cycle obtained by FACS for Calu-3 cells in 2D culture and 3D-printed at Time 0, after 24 and 72 h in culture, respectively. (B) Cell cycle for the culture obtained in (A) phases: G0/G1, S, G2/M. PI: propidium iodide (n = 3).

Figure 6

Nanoparticle diffusion in the uncontrolled deposition (unprinted) and printed OBST. Microscopical images on unprinted (A) and printed (B) hydrogel showing NP (green) and Calu-3 (red) interaction, respectively, in cell-laden multilayers (from top (0) to bottom (1500 µm)). Blue arrow: cell growth direction. (C) Left: a portion of a 3D-printed multilayer. Right: rendered image of the yellow zoom shown in the left panel. NP internalization from the cells. (D) Left: cell distribution in the unprinted hydrogel. Right: NP diffusion in the unprinted hydrogel. (E) Same as (D) for 3D-printed hydrogel.

Figure 7

Nanotoxicity exerted by silver nanoparticles in 3D-printed cell-laden multilayer. (A) Dose/response for Calu-3 exposed to Ag NPs (from 0 to 1000 ppm) in 2D environment for 48 h. (B) Same as (A) for the 3D-printed cells.