Solution: 3D cell culture methods that involve cell aggregation either by self-assembly into spheroids or the use of scaffolds to build tissue-like structures can enable long term experiments spanning a week or more. Peter Banks and Brad Larson of BioTek have shown that human hepatocytes demonstrate excellent plasma membrane integrity and enzyme activity (i.e. cytochrome P450, isotype 3A4) over a period of almost two weeks when contained in a spheroid liver microtissue. This allows for drug toxicity testing over the same period.
Gen5 software’s image analysis capability allows the images to be processed in many ways, providing greater detail on how diclofenac affects the spheroid. The mean green fluorescence in the field of view can be plotted (Figure 1B), providing a relative impression of increasing toxicity based on the disruption of the plasma membranes of the cells comprising the spheroid. A fluorescence ratio of green (dead cell fluorescence) to blue (total cell fluorescence) can also be plotted, which yields a different profile at higher diclofenac concentration. These data are equivalent to that obtainable from PMT-based optics, but with improved S/B which suggests that diclofenac toxicity reaches a maximum at 30 μM. Ratiometric data is rather confusing as it suggests that diclofenac toxicity is inexplicably reduced at higher concentration. Upon examining the spheroid images in Figure 1A, it is evident that the spheroids are expanding with increasing diclofenac concentration. Gen5 can use image analysis routines to compute spheroid size, based on a diameter and spheroid area which is plotted in Figure 1C. Quantitative data indicate that the spheroids do increase in size up to 30 μM diclofenac, but at the highest dose, it appears the integrity of the spheroid is partially ruptured and the size and area is reduced. These data suggest an explanation for the ratiometric data seen in Figure 1B. Finally, cell counting in a focal plane that bisects the spheroid can be performed for both blue (live cell) and green (dead cell) fluorescence channels using Gen5. Quantitative data presented in Figure 1D indicates that while the total number of cells counted in the spheroids is consistent across all diclofenac concentrations, increasing toxicity is evident across the concentration profile of diclofenac reaching almost 100 percent toxicity at 300 μM.
Fluorescence microscopy and the image analysis capabilities of Gen5 provide multiparametric data that allows for a complete understanding of long-term diclofenac toxicity on spheroid microtissues.
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