Efficient purification of CD4+ lymphocytes from peripheral blood progenitor cell products using affinity bead acoustophoresis

Abstract

Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-beadmediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n 5 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD41 lymphocytes were acoustically isolated with a mean (6SD) purity of 87 6 12%, compared with 96 6 3% for control magnetic sorting. Viability of sorted cells was 95 6 4% (acoustic) and 97 6 3% (magnetic), respectively. The mean acoustic separation efficiency of CD41 lymphocytes to the target fraction was 65 6 22%, compared with a mean CD41 lymphocyte recovery of 56 6 15% for magnetic sorting. Functional testing of targeted CD41 lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD41 lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinitybead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology. V C 2014 International Society for Advancement of CytometryKey terms peripheral blood progenitor cells; PBPC; lymphocytes; magnetic beads; acoustophoresis; ultrasound; cell sorting CELL separation technologies are essential tools in the processing of hematopoietic cells for therapeutic administration, diagnostic purposes or research applications. Separation by centrifugal force is generally employed for primary extraction of leukocytes from whole blood or bone marrow (1), followed by selective affinity methods, such as magnetic activated cell sorting (MACS) (2), or fluorescence activated cell sorting (FACS) (3), for isolation or depletion of specific cell populations. Depending on sample characteristics and the specific cell sorting application, the currently available techniques have distinct advantages as well as disadvantages in terms of recovery, purity, and throughput ...

Isolation and characterization of primary bone marrow mesenchymal stromal cells

“…Finally, novel label-free sorting techniques based on biophysical properties have been recently reported for the sorting of MSC subpopulations in culture. 61 Other alternatives are ultrasound-based techniques, which are currently being investigated in our laboratory 62,63 and which can possibly be applied to purify pre-and postculture clinical MSC products.…”

Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip

“…Furthermore, both piezo-actuators (trapping and levitation) were characterized by admittance analysis using an impedance analyzer (Z-Check 16777k, Sinephase, Austria), see Fig. 1A- [8][9]. Hence, both the resonance frequencies and Q-factors of the piezo-actuators were calculated from electrical impedance measurements, and were used as starting values before manual fine tuning of the driving frequency based on experimental observations.…”

Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques

“…Given the rarity of SSCs and the complexity of bone marrow tissue, none of these papers described a technology that would be suitable for SSC isolation. Finally, only one publication reports a high throughput (300,000 cells/min) (Lenshof et al, 2014), of the order of magnitude required for SSC isolation from a bone marrow buffy coat which can comprise up to a hundred million cells. Thus, acoustophoresis may not demonstrate the throughput required for sorting SSCs from bone marrow harvests.…”

“…The authors also demonstrated that the progenitor cells retained viability and maintained their colony-forming ability post-sorting. The same group used immunomagnetic beads to alter the acoustic properties of the cells to achieve improved separation (Lenshof et al, 2014). CD4 + lymphocytes were separated from RBC-depleted PBPC samples with 87% purity and separation efficiencies of 65%, comparable to MACS.…”

“…+ lymphocytes needed to be immuno-labelled to alter their acoustic properties and allow separation (Lenshof et al, 2014). Sorting SSCs based on size differences is unlikely to discriminate sufficiently, as SSCs are characterised by a broad distribution in size and co-localise mainly with the monocyte population (Janeczek et al, 2015b).…”