QUANTITATIVE RECORDING OF VITALITY PATTERNS IN LIVING MULTICELLULAR SPHEROIDS BY CONFOCAL MICROSCOPY

Fluorescent dyes were used in conjunction with confocal microscopy to record the vitality status of cells in multicellular glioma spheroids. Multicellular spheroids are in vitro models for micrometastases or in travascular microregions of large tumors. With progressing growth thre e distinct concentric annular shells develop. A rim of proliferating c ells in the periphery is followed towards the center by layers of quie scent cells and al a defined spheroid diameter cell death occurs in th e central core. Fluorescein diacetate (FDA) and Calcein/AM were used a s vital stains and Lucifer Yellow/VS (LYVS) was used as a marker for d ead cells. For loading multicellular spheroids with the esterase subst rate dyes we used a two step cold incubation technique to avoid dye ac cumulation in the most peripheral cell layers. Homogenously stained ti ssue allowed to describe the fluorescence attenuation in depth as a mo noexponential decay. An attenuation coefficient C was calculated from calibration experiments to be 12.5 . 10(-3) in vital stained tissue an d 17.9 . 10(-3) in lethal stained tissue. Using the respective attenua tion coefficient the raw data were corrected for light absorbtion and scattering in depth. In radial recordings of the vitality status of mu lticellular glioma spheroids using CLSM-technique we showed that spher oids up to a diameter of 250 mu m were homogenously stained with Calce in/AM and FDA. Spheroids larger than 250 mu m consist of vital stained cells and unstained cells. They do not show dead cell staining until they reach a diameter of about 400 mu m. The thickness of the rim of v ital stained cells decreased with increasing diameter of the spheroids to 64+/-7 mu m in spheroids of a diameter of 550+/-25 mu m. Thereafte r the thickness of the Calcein/AM or FDA stained rim augmented again, reaching 93+/-9 mu m in spheroids of 700 mu m in diameter. The first s igns of dead cell staining in the central core occurred at a diameter of 400+/-25 mu m. The radius of the core increased in an exponential w ay. The cell layer which was stained neither by vital nor by lethal dy es showed a thickness of 150 mu m in spheroids of 550+/-25 mu m in dia meter. Our staining technique and the radial recording of mean field f luorescence signals in living multicellular spheroids will be a valuab le tool for experimental cancer research providing a non invasive quan tification of cell vitality in living multicellular spheroids.