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.
Highlights
Assembly consisting of gold, iron oxide, and poly-L-lysine, after which they are directed using mild magnetic forces[18,19,20,21,22,23,24,25,26,27,28,29]
Image analysis showed that over the course of 10 h, the 3T3 spheroids contracted except when exposed to penicillin-G, the negative control (Fig. 2, see Supplemental Movie M1 for a movie of spheroid contraction in response to all-trans retinoic acid (ATRA))
Spheroid contraction is a unique endpoint in which viable cells rearrange and compact immediately after printing as a result of cell-cell interactions to contract the spheroid into a smaller size as a starting point for long-term growth (Fig. 1)
Summary
Assembly consisting of gold, iron oxide, and poly-L-lysine, after which they are directed using mild magnetic forces[18,19,20,21,22,23,24,25,26,27,28,29]. Similar methods have previously been employed to simulate such tissues as fat[23], lung[27], aortic valve[28], blood vessels[21,22], and tumor microenvironments, such as that of breast cancer[29] and glioblastoma[18,19], all of which show in vivo-like protein expression and ECM From this foundation, assays can be developed that take advantage of the representative environment of spheroids. With M3DB spheroids, building on the principles of the aforementioned ring assay, a novel assay was developed utilizing spheroid size and its change because of early events, like compaction[32,33,34], as an endpoint for cytotoxicity. The results of this study help demonstrate that this assay using M3DB spheroids can overcome the technical limitations in speed, throughput, handling, and imaging of other 3D cell culture platforms to offer a simple yet robust assay for the determination of general cytotoxicity in a 3D environment
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