Multitarget magnetic activated cell sorter

Abstract

Magnetic selection allows high-throughput sorting of target cells based on surface markers, and it is extensively used in biotechnology for a wide range of applications from in vitro diagnostics to cell-based therapies. However, existing methods can only perform separation based on a single parameter (i.e., the presence or absence of magnetization), and therefore, the simultaneous sorting of multiple targets at high levels of purity, recovery, and throughput remains a challenge. In this work, we present an alternative system, the multitarget magnetic activated cell sorter (MT-MACS), which makes use of microfluidics technology to achieve simultaneous spatially-addressable sorting of multiple target cell types in a continuous-flow manner. We used the MT-MACS device to purify 2 types of target cells, which had been labeled via target-specific affinity reagents with 2 different magnetic tags with distinct saturation magnetization and size. The device was engineered so that the combined effects of the hydrodynamic force produced from the laminar flow and the magnetophoretic force produced from patterned ferromagnetic structures within the microchannel result in the selective purification of the differentially labeled target cells into multiple independent outlets. We demonstrate here the capability to simultaneously sort multiple magnetic tags with >90% purity and >5,000-fold enrichment and multiple bacterial cell types with >90% purity and >500-fold enrichment at a throughput of 10(9) cells per hour.

Fig. 1.

MT-MACS separation architecture. (A) (Step A) The sample contains an excess of nontarget cells and 2 different target cells (target 1 and target 2) that are labeled with 2 different magnetic tags (tag 1 and tag 2) by specific surface markers. (Step B) The sample is continuously pumped into the device where the 2 target cell types are sorted into spatially-segregated independent outlets. Separation occurs in 2 regions of high magnetic field gradient generated by the microfabricated ferromagnetic strip (MFS) 1 and MFS 2. (Step C) After sorting, the eluted fractions from each outlet are analyzed via flow cytometry. (B) A free-body diagram showing the balance of forces at the MFS structures. At MFS 1 (θ₁ = 15°), tag 1-labeled target 1 cells are deflected and elute through outlet 1 because F_m1 > F_d1 sin(θ₁). This is not the case for tag 2-labeled target 2 cells, which are instead deflected at MFS 2 (θ₂ = 5°) because F_m2 > F_d2 sin(θ₂), and elute through outlet 2. Nontarget cells are not deflected by either MFS and elute through the waste outlet. (C) Optical micrographs (magnification = 100×) of the tags being separated at the 2 MFS structures at a total flow rate of 47 mL/h (sample = 5 mL/h, buffer = 42 mL/h). (Left) Tag 1 is deflected by the steep angled MFS 1. (Right) Tag 2 is deflected by MFS 2.

Fig. 2.

Numerical simulation of the long-range and short-range magnetic field gradients. (A) The long-range magnetic field gradient is generated by the external permanent magnets. The magnitude of the downward (−y direction) gradient (>200 T/m) extends over the full length of the main fluidic channel. (Inset) See Fig. S1 for position of the external magnets. (B) An abrupt change in relative permittivity (μ_r) between the microfabricated nickel features (μ_r ≈ 200) and the surrounding material (μ_r ≈ 1) creates large short-range magnetic field gradients in the vicinity of the MFS. The magnitude of this gradient is high (>10⁴ T/m) and it extends ≈8 μm from the MFS. The large magnetophoretic forces for target deflection are generated by these short-range gradients. The height of the image corresponds to the height of the fluidic channel.

Fig. 3.

Quantitative measurement of MT-MACS sorting performance for magnetic tags by flow cytometry. a.u., arbitrary units. (A) The initial sample consisted primarily of nontarget polystyrene beads (99.974%, r = 2.5 μm) and fractional percentages of tag 1 (0.020%, r = 2.25 μm) and tag 2 (0.006%, r = 1.4 μm). (B) After a single round of separation, the population of tag 1 recovered via outlet 1 was enriched to 95.876%, an ≈5,000-fold enrichment. (C) In outlet 2, the concentration of tag 2 was enriched ≈15,000-fold to 90.517%. (D) Material recovered at the waste outlet consisted almost entirely of nontarget beads, with only 0.002% tag 1 and 0.001% tag 2 in the eluted fraction.

Fig. 4.

Cytometric analysis of simultaneous multitarget bacterial cell sorting. (A) The initial sample mixture consists of 99.442% nontarget cells (expressing BFP) doped with 0.175% target 1 cells and 0.383% target 2 cells. In a single pass through the device, both targets were simultaneously enriched to >90% purity. (B) The cell mixture recovered at the target 1 outlet consisted of 91.575% target 1 cells, 8.393% target 2 cells, and 0.032% nontarget cells. (C) The output at the target 2 outlet comprised 93.865% target 2 cells, 6.123% target 1 cells, and 0.012% nontarget cells. (D) The output via the waste outlet consisted of 99.621% nontarget cells, 0.102% target 1 cells, and 0.277% target 2 cells.