High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array

TLDR

Three‑dimensional cell aggregates improve in vitro biological and therapeutic testing but are typically more complex, cumbersome, and costly than two‑dimensional cultures. The study aims to develop a 384‑well hanging‑drop plate that simplifies spheroid formation, culture, and drug testing for high‑throughput screening. The authors implement a 384‑well hanging‑drop plate that enables straightforward spheroid formation, culture, and drug testing compatible with standard high‑throughput screening instruments. The platform reveals that 5‑fluorouracil is more effective in 2D cultures, while tirapazamine preferentially kills 3D spheroids, demonstrating that the 384‑well hanging‑drop system.

Abstract

Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.