Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment

Abstract

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay

“…The spheroids are then transferred to the spheroid separation plate, ensuring their immobilization in a fixed position, a prerequisite for high-resolution imaging techniques like confocal imaging. 19 An additional advantage of the 3DHS-FP lies in the design of the spheroid separation plate. This component incorporates a hexagonal array of pillars and channels, each measuring 0.12 mm.…”

“…Previous methods for drug evaluation using spheroids typically involved additional assays such as colorimetric viability assays or live/dead staining, which were not only labour intensive but also incurred significant costs. 16,19 In contrast, with the 3DHS-FP, we can evaluate the efficacy of drugs solely based on changes in spheroid size, obviating the need for supplementary analyses. Notably, the 3DHS-FP surpasses the capabilities of our earlier 3D hanging drop plate, 17 providing enhanced simplicity and convenience by eliminating the requirement for tilting and enabling automatic separation of dead cells through gravitational forces.…”

“…2A and B). The 3D hanging dripper was designed with a centre inlet (2 mm in diameter) and a side inlet (1.5 mm in diameter) like the previously developed multi-inlet spheroid generator (MSG) 19 and fabricated by an 3D printer (Single Plus – 320C, Cubicon) with an ABS filament. It consists of five individual hanging drops connected in a row by lateral wings on both sides (2 × 2 × 4 mm 3 , width × length × height).…”