Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing

Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research

The primer sequences used for qRT-PCR analyses.

<p>The primer sequences used for qRT-PCR analyses.</p

Assessment of drug cytotoxicity on agarose scaffolds with micro-wells.

<p>Data were expressed as mean ± standard deviation (SD) (n = 6). (A) Cytotoxicity testing of doxorubicin against MCF-7 cells in 3-D and 2-D cell culture models. (B) Size-dependent drug responses in 3-D cell culture models.</p

Apoptosis of MCF-7 cells in spheroid culture and monolayer culture after 8 and 12 days of culture.

<p>*Statistical significance (P<0.05) between 2-D monolayer cells and 3-D sphere cells.</p><p>Apoptosis of MCF-7 cells in spheroid culture and monolayer culture after 8 and 12 days of culture.</p

IC₅₀ (μg/mL) and MCRI of systems assayed for drug resistance.

<p><i>P</i> values indicate significant differences between IC₅₀ values in 2-D and 3-D cultures.</p><p>IC₅₀ (μg/mL) and MCRI of systems assayed for drug resistance.</p

Penetration of DOX in MCF-7 spheroids.

<p>(A) Cross-sections at the middle of the spheroids were analyzed. (B) LSCM representative images of spheroid cross-sections, with different sizes, incubated with DOX for 0.5 h, 2 h, and 4 h. Scale bars, 100 μm. (C) Penetration area of DOX where the drug signals were detectable and normalized by area.</p

Generation of MCF-7 spheroids with different sizes by varying initial seeding cell concentrations.

<p>The diameters of spheroids were determined after 3 days of culture. (A) Spheroids with different diameters. Spheroid diameters of each group were highly uniform (SD ≤ 10%). Scale bars, 100 μm. (B) Spheroid size can be controlled by varying cell seeding number. Data represent the mean ± standard deviation (SD) from one experiment (n = 30).</p