A Three-Dimensional Cell Culture Platform for Long Time-Scale Observations of Bio–Nano Interactions

Abstract

We know surprisingly little about the long-term outcomes for nanomaterials interacting with organisms. To date, most of what we know is derived from in vivo studies that limit the range of materials studied, and the scope of advanced molecular biology tools applied. Long-term in vitro nanoparticle studies are hampered by a lack of suitable models, as standard cell culture techniques present several drawbacks, while technical limitations render current 3D cellular spheroid models less suited. Now, by controlling the kinetic processes of cell assembly and division in a non-Newtonian culture medium, we engineer reproducible cell clusters of controlled size and phenotype, leading to a convenient and flexible long-term 3D culture that allows nanoparticle studies over many weeks in an in vitro setting. We present applications of this model for the assessment of intracellular polymeric and silica nanoparticle persistence, and found that hydrocarbon based polymeric nanoparticles undergo no apparent degradation over long time periods with no obvious biological impact, while amorphous silica nanoparticles degrade at different rates over several weeks, depending on their synthesis method.

Quantifying the level of nanoparticle uptake in mammalian cells using flow cytometry

“…It is important to minimize the dilution of intracellular nanoparticles due to cell cycling, because the doubling time of most mammalian cell lines is longer than 24 h. Muraca et al employed a three-dimensional spheroid culture enabling the long-term monitoring of nanomaterials interacting with organisms, where cell cycling is effectively modulated and cells enter the quiescent (G0) phase. 50 A549 cells cultured under serum-free conditions for 24 h underwent morphological changes: they lost cell-cell contact and rolled up rather than spreading out on the culture dish. Cell proliferation was inhibited and fewer cells were observed via a micrograph even with the lowest tested nanoparticle concentration, 10 ng mL −1 FITC-SiO 2 NP25.…”

A Focus on “Bio” in Bio–Nanoscience: The Impact of Biological Factors on Nanomaterial Interactions

“…In addition, control of the assembly process of 3D models can allow for long‐term studies of bio–nano interactions over many weeks, potentially leading to a more accurate assessment of the long‐term outcomes for particle interactions with biological systems, including degradation properties of nanomaterials. [ 136 ]…”

“…All phases of the cell cycle are represented in spheroids, although in models of quiescent cells, cells in G0 are dominant. [ 11,136 ] The effect of the cell cycle in the context of drug delivery in spheroids has been reviewed. [ 11 ] As in 2D cells, cells in distinct growth phases (the cell cycle status) in 3D models might differ in how they respond to NPs and therapeutics, particularly with gene delivery systems, which may be highly cell cycle dependent.…”