Viable sorting of intact multicellular spheroids by flow cytometry

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Abstract
A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly‐sized populations with diameters in the range of 50–100 μm. A FACS II instrument was modified for this purpose by installing a 200‐μm‐diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 μm diameter) and 41–96‐μm‐diameter spheroids could be sorted and recovered with 70–100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small‐angle forward light scatter, 90° light scatter, and autofluorescence using a 488‐nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90° light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90° light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5–9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as in investigations of the contact effect or in the initiation of growth curve studies.