Viable sorting of intact multicellular spheroids by flow cytometry.

Area of Science:

• Biotechnology
• Cell Biology
• Biophysics

Background:

• Multicellular spheroids are valuable models in cancer research and drug development.
• Obtaining uniformly sized spheroid populations is crucial for reproducible experimental results.
• Previous spheroid sorting methods lacked efficiency and precision.

Purpose of the Study:

• To develop and validate a flow cytometric method for sorting viable, intact multicellular spheroids.
• To achieve uniformly sized spheroid populations within a specific diameter range (50-100 microns).
• To improve the efficiency and precision of spheroid sorting compared to existing techniques.

Main Methods:

• Modification of a Fluorescence-Activated Cell Sorting (FACS) II instrument with a 200-micron exit orifice.
• Optimization of sheath flow, oscillator frequency, and droplet sorting parameters.
• Simultaneous analysis of forward light scatter, 90-degree light scatter, and autofluorescence using a 488-nm laser.

Main Results:

• Successful sorting and recovery of polystyrene microspheres and spheroids with 70-100% efficiency.
• Development of sort windows based on forward light scatter, 90-degree light scatter, and autofluorescence to isolate uniform spheroid populations.
• Achieved coefficient of variation for mean spheroid diameter between 5-9%, representing less than one cell diameter variation.
• Demonstrated no significant difference in the subsequent growth rates of sorted versus unsorted spheroids.

Conclusions:

• The developed flow cytometric method provides a highly efficient and precise way to sort viable multicellular spheroids by size.
• This technique yields exceptionally uniform spheroid populations, a significant advancement over existing methods.
• The method is applicable to various research areas requiring uniform spheroid populations, such as contact effect studies and growth curve initiation.