Response to Drug Treatment-Cell Cycle Analysis of 3D Cell Cultures

The effect of anticancer drugs on 3D cell cultures can be evaluated through cell imaging.
In combination with cell-cycle indicator Fucci, the effect of an anticancer drug on the cell cycle can be investigated.

Objectives

Recently, in addition to conventional 2D cell cultures, 3D cell cultures were used in the mechanistic study of anticancer drugs. In this study, the response of 3D cell cultures to drugs was analyzed in terms of cell cycle as well as cell death.

Preparation of samples

Fucci is a cell-cycle indicator using a dual color scheme. A cell suspension of Fucci expressing transgenic HT-29 cells was seeded into a U-bottom 96-well plate. Cell cultures were exposed to serially diluted cisplatin (0-10 nM) 72 hours after the start of cell culture. After incubating for 96 hours, the medium containing cisplatin was removed from the cell cultures, and they were fixed in 4% formalin.

Conclusion
Acquisition and analysis of fluorescent images

Confocal fluorescent images of 3D cell cultures were obtained through the combined use of the cell imaging analysis system (Ex: 488 nm and 561 nm). Fluorescence luminosity data were treated with maximum intensity projection followed by the identification of nuclei with analysis software and cell counting. The number of HT-29 cells in spheroids 96 hours after the addition of cisplatin are shown in Figure 1. The analysis of dual color signaling indicated that the treatment of 3D cell cultures  with cisplatin resulted in the reduction of G0 and G1 phases of the cell cycle as well as the cell number (Figures 2 and 3).

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