Purification of regulatory T cells with the use of a fully enclosed high-speed microfluidic system.

Abstract

Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4(+)/CD25(bright)/CD127(neg/low). The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-μm polystyrene particles. CD4(+)-enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel microfluidic-based sorters. Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability.