Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Acoustofluidic Properties of Polystyrene Microparticles.

Acoustophoresis has become a powerful tool to separate microparticles and cells, based on their material and biophysical properties, and is gaining popularity in clinical and biomedical research. One major application of acoustophoresis is to measure the compressibility of cells and small organisms, which is related to their contents. The cell compressibility can be extracted from the acoustic mobility, which is the main output of acoustic migration experiments, if the material properties and sizes of reference particles, the size of the cells, and the surrounding medium are known. Accurate methods to measure and calibrate the acoustic energy density in acoustophoresis systems are therefore critical. In this Perspective, polystyrene microparticles have become the most commonly used reference particles in acoustophoresis, due to their similar biophysical properties to cells. We utilized a two-step focusing method to measure the relative acoustic mobility of polystyrene beads of various sizes and colors and present a quantitative analysis of the variation in acousto-mechanical properties of polystyrene microparticles, showing a large spread in their material properties. A variation of more than 25% between different particle types was found. Thus, care is required when relying on polystyrene particles as a reference when characterizing acoustofluidics systems or acousto-mechanical properties of cells.

Advancement and obstacles in microfluidics-based isolation of extracellular vesicles.

There is a great need for techniques which enable reproducible separation of extracellular vesicles (EVs) from biofluids with high recovery, purity and throughput. The development of new techniques for isolation of EVs from minute sample volumes is instrumental in enabling EV-based biomarker profiling in large biobank cohorts and paves the way to improved diagnostic profiles in precision medicine. Recent advances in microfluidics-based devices offer a toolbox for separating EVs from small sample volumes. Microfluidic devices that have been used in EV isolation utilise different fundamental principles and rely largely on benefits of scaling laws as the biofluid processing is miniaturised to chip level. Here, we review the progress in the practicality and performance of both passive devices (such as mechanical filtering and hydrodynamic focusing) and active devices (using magnetic, electric or acoustic fields). As it stands, many microfluidic devices isolate intact EV populations at higher purities than centrifugation, precipitation or size-exclusion chromatography. However, this comes at a cost. We address challenges (in particular low throughput, clogging risks and ability to process biofluids) and highlight the need for more improvements in microfluidic devices. Finally, we conclude that there is a need to refine and standardise these lab-on-a-chip techniques to meet the growing interest in the diagnostic and therapeutic value of purified EVs.

Acoustofluidic platforms for particle manipulation.

There is an increasing interest in acoustics for microfluidic applications. This field, commonly known as acoustofluidics involves the interaction of ultrasonic standing waves with fluids and dispersed microparticles. The combination of microfluidics and the so-called acoustic standing waves (ASWs) led to the development of integrated systems for contact-less on-chip cell and particle manipulation where it is possible to move and spatially localize these particles based on the different acoustophysical properties. While it was initially suggested that the acoustic forces could be harmful to the cells and could impact cell viability, proliferation, or function via phenotypic or even genotypic changes, further studies disproved such claims. This review is summarizing some interesting applications of acoustofluidics in the manipulations of biomaterials, such as cells or subcellular vesicles, in works published mainly within the last 5 years.

Two-Step Acoustophoresis Separation of Live Tumor Cells from Whole Blood.

There is an unmet clinical need to extract living circulating tumor cells (CTCs) for functional studies and in vitro expansion to enable drug testing and predict responses to therapy in metastatic cancer. Here, we present a novel two-step acoustophoresis (A2) method for isolation of unfixed, viable cancer cells from red blood cell (RBC) lysed whole blood. The A2 method uses an initial acoustofluidic preseparation step to separate cells based on their acoustic mobility. This acoustofluidic step enriches viable cancer cells in a central outlet, but a significant number of white blood cells (WBCs) remain in the central outlet fraction due to overlapping acoustophysical properties of these viable cells. A subsequent purging step was employed to remove contaminating WBCs through negative selection acoustophoresis with anti-CD45-functionalized negative acoustic contrast particles. We processed 1 mL samples of 1:1 diluted RBC lysed whole blood mixed with 10 000 DU145 cells through the A2 method. Additional experiments were performed using 1000 DU145 cells spiked into 1.5 × 106 WBCs in 1 mL of buffer to further elucidate the dynamic range of the method. Using samples with 10 000 DU145 cells, we obtained 459 ± 188-fold depletion of WBC and 42% recovery of viable cancer cells. Based on spiked samples with 1000 DU145 cells, our cancer cell recovery was 28% with 247 ± 156-fold WBC depletion corresponding to a depletion efficacy of ≥99.5%. The novel A2 method provides extensive elimination of WBCs combined with the gentle recovery of viable cancer cells suitable for downstream functional analyses and in vitro culture.

Multinodal Acoustic Trapping Enables High Capacity and High Throughput Enrichment of Extracellular Vesicles and Microparticles in miRNA and MS Proteomics Studies.

We report a new design of an acoustophoretic trapping device with significantly increased capacity and throughput, compared to current commercial acoustic trapping systems. Acoustic trapping enables nanoparticle and extracellular vesicle (EV) enrichment without ultracentrifugation. Current commercial acoustic trapping technology uses an acoustic single-node resonance and typically operates at flow rates <50 µL/min, which limits the processing of the larger samples. Here, we use a larger capillary that supports an acoustic multinode resonance, which increased the seed particle capacity 40 times and throughput 25-40 times compared to single-node systems. The resulting increase in capacity and throughput was demonstrated by isolation of nanogram amounts of microRNA from acoustically trapped urinary EVs within 10 min. Additionally, the improved trapping performance enabled isolation of extracellular vesicles for downstream mass spectrometry analysis. This was demonstrated by the differential protein abundance profiling of urine samples (1-3 mL), derived from the non-trapped versus trapped urine samples.

Clinical-Scale Cell-Surface-Marker Independent Acoustic Microfluidic Enrichment of Tumor Cells from Blood.

Enumeration of circulating tumor cells (CTCs) predicts overall survival and treatment response in metastatic cancer, but as many commercialized assays isolate CTCs positive for epithelial cell markers alone, CTCs with little or no epithelial cell adhesion molecule (EpCAM) expression stay undetected. Therefore, CTC enrichment and isolation by label-free methods based on biophysical rather than biochemical properties could provide a more representative spectrum of CTCs. Here, we report on a clinical-scale automated acoustic microfluidic platform processing 5 mL of erythrocyte-depleted paraformaldehyde (PFA)-fixed blood (diluted 1:2) at a flow rate of 75 µL/min, recovering 43/50 (86 ± 2.3%) breast cancer cell line cells (MCF7), with 0.11% cancer cell purity and 162-fold enrichment in close to 2 h based on intrinsic biophysical cell properties. Adjustments of the voltage settings aimed at higher cancer cell purity in the central outlet provided 0.72% cancer cell purity and 1445-fold enrichment that resulted in 62 ± 8.7% cancer cell recovery. Similar rates of cancer-cell recovery, cancer-cell purity, and fold-enrichment were seen with both prostate cancer (DU145, PC3) and breast cancer (MCF7) cell line cells. We identified eosinophil granulocytes as the predominant white blood cell (WBC) contaminant (85%) in the enriched cancer-cell fraction. Processing of viable cancer cells in erythrocyte-depleted blood provided slightly reduced results as to fixed cells (77% cancer cells in the enriched cancer cell fraction, with 0.2% WBC contamination). We demonstrate feasibility of enriching either PFA-fixed or viable cancer cells with a clinical-scale acoustic microfluidic platform that can be adjusted to meet requirements for either high cancer-cell recovery or higher purity and can process 5 mL blood samples in close to 2 h.

Acoustophoretic removal of proteins from blood components.

This work presents the development of a miniaturized system for removing plasma proteins and other low-molecular-weight compounds from red blood cell (RBC) concentrate in a simple one-step-process using integrated ultrasound. The technology utilizes the principles of acoustophoresis to transfer the RBCs from the original plasma-containing solution into a protein-free SAG-M additive solution in a continuous flow process. The preparation of protein free RBC concentrate is important for blood transfusion to patients suffering from immunoglobulin A (IgA)-deficiency and developing antibodies against IgA. We show a nearly complete removal of both albumin and IgA from concentrated RBCs via this one-step-processes in samples obtained from RBC concentrate. The cell recovery of our technology is close to 97%, compared to just above 90% of the current procedure of repeated dilution and centrifugation steps. This work clearly shows the potential of integrated acoustophoresis in a miniaturized system for clinical applications.

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

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-bead-mediated 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 = 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 CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ 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 CD4+ 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 affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology. © 2014 International Society for Advancement of Cytometry.

Ultrasound standing wave spatial patterning of human umbilical vein endothelial cells for 3D micro-vascular networks formation.

Generating functional and perfusable micro-vascular networks is an important goal for the fabrication of large and three-dimensional tissues. Up to now, the fabrication of micro-vascular networks is a complicated multitask involving several different factors such as time consuming, cells survival, micro-diameter vasculature and strict alignment. Here, we propose a technique combining multi-material extrusion and ultrasound standing wave forces to create a network structure of human umbilical vein endothelial cells within a mixture of calcium alginate and decellularized extracellular matrix. The functionality of the matured microvasculature networks was demonstrated through the enhancement of cell-cell adhesion, angiogenesis process, and perfusion tests with microparticles, FITC-dextran, and whole mouse blood. Moreover, animal experiments exhibited the implantability including that the pre-existing blood vessels of the host sprout towards the preformed vessels of the scaffold over time and the microvessels inside the implanted scaffold matured from empty tubular structures to functional blood-carrying microvessels in two weeks.

Acoustic enrichment of heterogenous circulating tumor cells and clusters from patients with metastatic prostate cancer.

Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Continuous separation of cells and particles in microfluidic systems.

The progress in microfabrication and lab-on-a-chip technologies has been a major area of development for new approaches to bioanalytics and integrated concepts for cell biology. Fundamental advances in the development of elastomer based microfluidics have been driving factors for making microfluidic technology available to a larger scientific community in the past years. In line with this, microfluidic separation of cells and particles is currently developing rapidly where key areas of interest are found in designing lab-on-a-chip systems that offer controlled microenvironments for studies of fundamental cell biology. More recently industrial interests are seen in the development of micro chip based flow cytometry technology both for preclinical research and clinical diagnostics. This critical review outlines the most recent developments in microfluidic technology for cell and particle separation in continuous flow based systems. (130 references).

Acoustic Cell Patterning in Hydrogel for Three-Dimensional Cell Network Formation.

In the field of engineered organ and drug development, three-dimensional network-structured tissue has been a long-sought goal. This paper presents a direct hydrogel extrusion process exposed to an ultrasound standing wave that aligns fibroblast cells to form a network structure. The frequency-shifted (2 MHz to 4 MHz) ultrasound actuation of a 400-micrometer square-shaped glass capillary that was continuously perfused by fibroblast cells suspended in sodium alginate generated a hydrogel string, with the fibroblasts aligned in single or quadruple streams. In the transition from the one-cell stream to the four-cell streams, the aligned fibroblast cells were continuously interconnected in the form of a branch and a junction. The ultrasound-exposed fibroblast cells displayed over 95% viability up to day 10 in culture medium without any significant difference from the unexposed fibroblast cells. This acoustofluidic method will be further applied to create a vascularized network by replacing fibroblast cells with human umbilical vein endothelial cells.

Acoustic whole blood plasmapheresis chip for prostate specific antigen microarray diagnostics.

The generation of high quality plasma from whole blood is of major interest for many biomedical analyses and clinical diagnostic methods. However, it has proven to be a major challenge to make use of microfluidic separation devices to process fluids with high cell content, such as whole blood. Here, we report on an acoustophoresis based separation chip that prepares diagnostic plasma from whole blood linked to a clinical application. This acoustic separator has the capacity to sequentially remove enriched blood cells in multiple steps to yield high quality plasma of low cellular content. The generated plasma fulfills the standard requirements (<6.0 x 10(9) erythrocytes/L) recommended by the Council of Europe. Further, we successfully linked the plasmapheresis microchip to our previously developed porous silicon sandwich antibody microarray chip for prostate specific antigen (PSA) detection. PSA was detected by good linearity (R(2) > 0.99) in the generated plasma via fluorescence readout without any signal amplification at clinically relevant levels (0.19-21.8 ng/mL).

Label-free separation of leukocyte subpopulations using high throughput multiplex acoustophoresis.

Multiplex separation of mixed cell samples is required in a variety of clinical and research applications. Herein, we present an acoustic microchip with multiple outlets and integrated pre-alignment channel to enable high performance and label-free separation of three different cell or particle fractions simultaneously at high sample throughput. By implementing a new cooling system for rigorous temperature control and minimal acoustic energy losses, we were able to operate the system isothermally and sort suspensions of 3, 5 and 7 µm beads with high efficiencies (>95.4%) and purities (>96.3%) at flow rates up to 500 µL min-1 corresponding to a throughput of ∼2.5 × 106 beads per min. Also, human viable white blood cells were successfully fractionated into lymphocytes, monocytes and granulocytes with high purities of 96.5 ± 1.6%, 71.8 ± 10.1% and 98.8 ± 0.5%, respectively, as well as high efficiencies (96.8 ± 3.3%, 66.7 ± 3.2% and 99.0 ± 0.7%) at flow rates up to 100 µL min-1 (∼100 000 cells per min). By increasing the flow rate up to 300 µL min-1 (∼300 000 cells per min) both lymphocytes and granulocytes were still recovered with high purities (92.8 ± 1.9%, 98.2 ± 1 .0%), whereas the monocyte purity decreased to 20.9 ± 10.3%. The proposed isothermal multiplex acoustophoresis platform offers efficient fractionation of complex samples in a label-free and continuous manner at thus far unreached high sample throughput rates.

Differential impedance spectra analysis reveals optimal actuation frequency in bulk mode acoustophoresis.

This work reports a method to select the optimal working frequency in transversal bulk resonator acoustophoretic devices by electrical impedance measurements. The impedance spectra of acoustophoretic devices are rich in spurious resonance peaks originating from different resonance modes in the system not directly related to the channel resonance, why direct measurement of the piezoelectric transducer impedance spectra is not a viable strategy. This work presents, for the first time, that the resonance modes of microchip integrated acoustophoresis channels can be identified by sequentially measuring the impedance spectra of the acoustophoretic device when the channel is filled with two different fluids and subsequently calculate the Normalized Differential Spectrum (NDS). Seven transversal bulk resonator acoustophoretic devices of different materials and designs were tested with successful results. The developed method enables a rapid, reproducible and precise determination of the optimal working frequency.

Acoustophoresis in wet-etched glass chips.

Acoustophoresis in microfluidic structures has primarily been reported in silicon microfabricated devices. This paper demonstrates, for the first time, acoustophoresis performed in isotropically etched glass chips providing a performance that matches that of the corresponding silicon microdevices. The resonance mode characteristics of the glass chip were equal to those of the silicon chip at its fundamental resonance. At higher order resonance modes the glass chip displays resonances at lower frequencies than the silicon chip. The cross-sectional profiles of acoustically focused particle streams are also reported for the first time, displaying particles confined in a vertical band in the channel center for both glass and silicon chips. A particle extraction efficiency of 98% at flow rates up to 200 microL/min (2% particle concentration) is reported for the glass chip at the fundamental resonance. The glass and silicon chips displayed equal particle extraction performance when tested for increasing particle concentrations of 2-15%, at a flow velocity of 12.9 cm/s for the glass chip and 14.8 cm/s for the silicon chip.

Rapid and effective enrichment of mononuclear cells from blood using acoustophoresis.

Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 105 cells/s, corresponding to 5 µl/min undiluted whole blood equivalent. Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques.

Affinity-Bead-Mediated Enrichment of CD8+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Acoustophoresis.

Acoustophoresis is a technique that applies ultrasonic standing wave forces in a microchannel to sort cells depending on their physical properties in relation to the surrounding media. Cell handling and separation for research and clinical applications aims to efficiently separate specific cell populations. Here, we investigated the sorting of CD8 lymphocytes from peripheral blood progenitor cell (PBPC) products by affinity-bead-mediated acoustophoresis. PBPC samples were obtained from healthy donors (n = 4) and patients (n = 18). Mononuclear cells were labeled with anti-CD8-coated magnetic beads and sorted on an acoustophoretic microfluidic device and by standard magnetic cell sorting as a reference method. CD8 lymphocytes were acoustically sorted with a mean purity of 91% ± 8% and a median separation efficiency of 63% (range 15.1%⁻90.5%) as compared to magnetic sorting (purity 91% ± 14%, recovery 29% (range 5.1%⁻47.3%)). The viability as well as the proliferation capacity of sorted lymphocytes in the target fraction were unimpaired and, furthermore, hematopoietic progenitor cell assay revealed a preserved clonogenic capacity post-sorting. Bead-mediated acoustophoresis can, therefore, be utilized to efficiently sort less frequent CD8+ lymphocytes from PBPC products in a continuous flow mode while maintaining cell viability and functional capacity of both target and non-target fractions.

Microchannel acoustophoresis does not impact survival or function of microglia, leukocytes or tumor cells.

BACKGROUND: The use of acoustic forces to manipulate particles or cells at the microfluidic scale (i.e. acoustophoresis), enables non-contact, label-free separation based on intrinsic cell properties such as size, density and compressibility. Acoustophoresis holds great promise as a cell separation technique in several research and clinical areas. However, it has been suggested that the force acting upon cells undergoing acoustophoresis may impact cell viability, proliferation or cell function via subtle phenotypic changes. If this were the case, it would suggest that the acoustophoresis method would be a less useful tool for many cell analysis applications as well as for cell therapy. METHODS: We investigate, for the first time, several key aspects of cellular changes following acoustophoretic processing. We used two settings of ultrasonic actuation, one that is used for cell sorting (10 Vpp operating voltage) and one that is close to the maximum of what the system can generate (20 Vpp). We used microglial cells and assessed cell viability and proliferation, as well as the inflammatory response that is indicative of more subtle changes in cellular phenotype. Furthermore, we adapted a similar methodology to monitor the response of human prostate cancer cells to acoustophoretic processing. Lastly, we analyzed the respiratory properties of human leukocytes and thrombocytes to explore if acoustophoretic processing has adverse effects. RESULTS: BV2 microglia were unaltered after acoustophoretic processing as measured by apoptosis and cell turnover assays as well as inflammatory cytokine response up to 48 h following acoustophoresis. Similarly, we found that acoustophoretic processing neither affected the cell viability of prostate cancer cells nor altered their prostate-specific antigen secretion following androgen receptor activation. Finally, human thrombocytes and leukocytes displayed unaltered mitochondrial respiratory function and integrity after acoustophoretic processing. CONCLUSION: We conclude that microchannel acoustophoresis can be used for effective continuous flow-based cell separation without affecting cell viability, proliferation, mitochondrial respiration or inflammatory status.