Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids

Abstract

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments.

Doxorubicin-sensitive and -resistant colorectal cancer spheroid models: assessing tumor microenvironment features for therapeutic modulation

“…Dox-sensitive (HCT116) and Dox-resistant (HCT116-DoxR) spheroids follow a similar global pattern of condensation regardless of the presence (heterotypic spheroids) or absence of fibroblasts (homotypic spheroids) ( Figures 2B ). There are some small differences: HCT116 spheroids condense as a whole ( Figures 2B ; Supplementary Video S1 ), showing a small contraction of their volume from 15 to 2.5 × 10 8 μm 3 , as previously demonstrated by Tartagni et al, 2023 , whereas, HCT116-DoxR spheroids form several small cell aggregates close to each other, that overtime condense with less variation of their total volume (approximately from 12 to 5.8 × 10 8 μm 3 ) ( Supplementary Video S2 ).…”

Recapitulating the Drifting and Fusion of Two-Generation Spheroids on Concave Agarose Microwells

“…Spheroids undergo multiple levels of self-assembly in the process of fusion. The size of the agarose-based micro-well is 2 mm in diameter and 2 mm in depth, thus having a larger volume than the agarose micro-wells used in previous studies [33,37,38]. The larger size of the agarose micro-well makes it possible to precisely pipette the same amount of cell suspension into each well, ensuring cell number uniformity between the wells and the reproducibility of different batches.…”