Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting.

Area of Science:

• Cell Biology
• Biotechnology
• Immunology

Background:

• Efficient isolation of specific cell subpopulations is crucial for both basic research and clinical applications.
• Common cell sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) have underreported performance metrics.
• Quantifying throughput, yield, viability, and processing time is essential for method selection.

Purpose of the Study:

• To quantitatively compare the performance of MACS and FACS for isolating alkaline phosphatase liver/bone/kidney (ALPL)-expressing and non-expressing cells.
• To assess the accuracy, cell loss, processing time, and viability associated with each sorting method.
• To determine optimal conditions for MACS to achieve accurate cell separation.

Main Methods:

• Defined mixtures of ALPL-expressing and non-expressing cells were sorted using both MACS and FACS.
• MACS sorting was performed using manufacturer's recommended reagent concentrations and optimized higher concentrations.
• Performance metrics including accuracy of cell splits, cell loss, processing time, and cell viability were recorded for both methods.

Main Results:

• Initial MACS runs with standard reagent concentrations yielded inaccurate cell separation when ALPL+ cells were proportionally high (>~25%).
• Substantially higher concentrations of labeling reagents were required for MACS to achieve accuracy comparable to FACS across all proportions.
• MACS sorting resulted in significantly lower cell loss (7-9%) compared to FACS (~70%) and was faster overall, especially for multiple samples processed in parallel.
• Cell viability remained high (>83%) for both methods across all experimental groups.

Conclusions:

• MACS requires optimization, particularly in reagent concentrations, to achieve accurate cell sorting across varying cell proportions.
• Despite optimization needs, MACS offers significant advantages in terms of reduced cell loss and faster processing times compared to FACS.
• The enhanced cell yield and speed of MACS make it a potentially valuable tool for translational and cell-based applications requiring large numbers of isolated cells.