A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging

Abstract

An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…Immediately afterwards, a magnet is placed on top of the plate lid (in magnetic levitation) or beneath the plate (in magnetic bioprinting), during which the SPION labeled cells are pulled up or down respectively under magnetic forces. The cells self-aggregate into spheroids within few hours (Tseng et al , 2015; Leonard et al , 2016) (Figure 3G). …”

Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment

“…Similarly, to produce tumour spheroids by avoiding cell adhesion to cultureware and inducing aggregation, an aqueous two-phase system can also compartmentalize cell suspension and produce spheroids without the concern of drying and possible inefficiency in chemical transport and toxicity of an oil phase [51][52][53]. Three-dimensional spheroids can also be formed by assembly of cells using bio-orthogonal chemistry [54] or incubation of cells with magnetic particles [55,56] (figure 2g,h). Recently, several microfluidic techniques have been developed to create tumour spheroids by either hydrodynamic trapping of cells in stagnation regions or in microwell structures [57][58][59][60], aggregating multiple cells in double-emulsions or hydrogel droplets [61][62][63][64], or aggregating cells on a digital microfluidic platform [65] (figure 2i).…”

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

“…Another successful example is the magnetic nanobeads-mediated spheroid formation [41][42][43][44][45][46][47][48][49] . This method (also known as 'magnetic levitation' when the cells are collected on top of the fluid in a tissue culture well rather than on its bottom 41 ), is extremely versatile.…”