A simple and rapid assay of lysosomal-targeting CDy6 for long-term real-time viability assessments in 2D and 3D in vitro culture models

Abstract

Abstract CDy6, a BODIPY-derived compound, is used to label lysosomes and visualize mitotic and proliferating cells. However, its effectiveness in long-term, real-time cell viability assays using both 2D and 3D cell culture models is unclear. Here we evaluated the suitability of CDy6 by assessing living cell viability and proliferation in HaCaT keratinocyte and CCD-986sk human fibroblast cell lines in 2D and 3D cell culture models. Cells were stained with CDy6 or other dyes and imaged using confocal microscopy to obtain fluorescence images. To analyze the absorbance of CDy6-targeted lysosomal vesicles (CLVs) derived from living cells, DMSO was added to the CDy6-stained HaCaT cells and then incubated for 1 hour at room temperature, and their absorbance was measured using a spectrometer. In addition, we tested the effects of CLVs on 3D cell culture models by adding CDy6-stained collagen hydrogels to CCD-986sk cells and loading them into a frame construction to establish a 3D dermal layer for long-term culture. The CDy6-based method, measured using a spectrometer, yielded results similar to those of the widely used MTT assay for measuring cell viability. Compared to calcein AM staining, the CLV method allows for both absorbance measurement and imaging under short-term and long-term culture conditions and resulted in less cytotoxicity. In conclusion, the CLV method provides a simple and sensitive tool for assessing the status of living cells in 2D and 3D cell culture models and can be used as an alternative to animal testing. Moreover, it is effective for monitoring cell viability under long-term real-time conditions in vitro.