Development of multicellular tumor spheroid (MCTS) culture from breast cancer cell and a high throughput screening method using the MTT assay

Abstract

In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures' response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.

Figure 1. Muticellular tumor spheroidal culture of MCF-7 with homogenous sizes.

The cells were cultured in a 96-well plate that was coated with agar gelrite. Cell aggregation was facilitated by centrifugation without the addition of any extracellular matrix to induce the formation of spheroid (Magnification: 1x, scale bar: 1 cm).

Figure 2. Morphological appearance of MCTS culture of MCF-7.

(A) With the facilitation by centrifugal force, MCF-7 cells were organized into a three-dimensional multicellular spheroidal structure. Under phase contrast microscope, the structure appeared to be compact and the cells were rigidly integrated into the solid structure where individual cells were indistinguishable from each other (magnification: 40x, scale bar: 100 µm). (B) Appearance of the culture under scanning electron microscopy (magnification: 160x, scale bar: 100 µm). (C) Single cells within the spheroid culture were connected to adjacent cells through cell-cell junction (arrow), which were responsible for the densely packed organization of the cells (magnification: 4000x, scale bar: 5 µm).

Figure 3. Viability of the monolayer and MCTS cultures of MCF-7 determined from the MTT assay.

Monolayer and MCTS cultures of the MCF-7 cell line were exposed to tamoxifen for 4 days. MTT assay for the MCTS cultures was carried out with some modification to the standard protocol by Mosmann (1983) while assay for the monolayer culture was performed in accordance to the original method.

Figure 4. Cytotoxicity of tamoxifen citrate against monolayer and MCTS cultures of MCF-7.

Cytotoxicity was determined by quantifying the percentage of LDH release relative to the maximum LDH release of untreated spheroid cultures. The results were presented as means ± S.E.M. of three independent experiments.

Figure 5. Comparison of the cytotoxicity increment in MCTS cultures assessed by MTT and LDH release assays.

Relative to the untreated control, which was normalized to 0% in both assays, reduction in cell viability of treated samples assessed by MTT assay was compared to the elevation in cytotoxicity determined from the LDH release assay. The results were presented as means ± S.E.M. of three independent experiments.