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Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors.

ABSTRACT: Three-dimensional (3D) tumor culture techniques are gaining popularity as in vitro models of tumoral tissue analogues. Despite the widespread interest, need, and present-day effort, most of the 3D tumor culturing methodologies have not gone beyond the inventors' laboratories. This, in turn, limits their applicability and standardization. In this study, we introduce a straightforward and user-friendly approach based on standard 96-well plates with basic amenities for growing 3D tumors in a scaffold-free/scaffold-based format. Hanging drop preparation can be easily employed by flipping a universal 96-well plate. The droplets of the medium generated by the well-plate flip (WPF) method can be easily modified to address various mechanisms and processes in cell biology, including cancer. To demonstrate the applicability and practicality of the conceived approach, we utilized human colorectal carcinoma cells (HCT116) to first show the generation of large scaffold-free 3D tumor spheroids over 1.5 mm in diameter in single-well plates. As a proof-of-concept, we also demonstrate matrix-assisted tumor culture techniques in advancing the broader use of 3D culture systems. The conceptualized WPF approach can be adapted for a range of applications in both basic and applied biological/engineering research.

SUBMITTER: Jeong Y

PROVIDER: S-EPMC9289396 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core-shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm-2), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 1010 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.

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Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors.

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