Buoyant microbubbles as alternatives or adjuncts to magnetic bead methods for high-purity cell isolation

Abstract

Abstract Widely used in immunologic applications, magnetic bead systems have significant limitations including cumbersome workflows, generation of high-shear and high-compressive forces during routine use, and limited performance in low and high volume samples. Buoyancy-activated cell sorting (BACS) overcomes these issues by using low-density microbubbles in lieu of magnets and iron beads to perform isolation. Here we report methods for three common uses: removal of extraneous RBCs prior to FACS, isolation of high-purity B lymphocytes from murine spleens, and improvement of a magnetic NK separation system by jointly applying BACS to remove contaminating NKT cells. RBC removal from leukopheresis-derived PBMC was performed with anti-CD235a-conjugated microbubbles. B cell isolation was performed with streptavidin microbubbles and a negative selection cocktail of biotinylated anti-murine antibodies. NK cell isolation was performed on PBMC using Miltenyi human CD56 MicroBeads and an additional purification step using streptavidin microbubbles and an anti-CD3 antibody. Each experiment used Akadeum microbubbles and a one-minute trituration to combine microbubbles with cell suspensions. RBC microbubbles provided 98.3 ± 1.0% removal of RBCs with 11.3 ± 0.9% nonspecific binding. B cells isolated with microbubbles were 91.8 ± 2.8% pure with a yield of 94.2 ± 4.3%. NK purification with magnetic microparticles produced 80.5 ± 0.7% purity. Additional purification with microbubbles increased purity to 90.8 ± 1.3%. Microbubbles are a flexible and simpler alternative to magnetic cell isolation systems, delivering comparable performance and, when used in conjunction with magnetic beads, superior purity.