RESUMEN

The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion. KEY POINTS: • Acoustic focusing in a microchannel was used to concentrate cell suspensions • The parameters influencing focusing at high concentrations were studied • Three different cell strains were successfully concentrated.

Asunto(s)

RESUMEN

Ultrasonic standing waves with specific wavelengths generated in the multi-layered micro-resonators were numerically and experimentally analyzed. Using a three-dimensional scanning fluorescence microscope, the acoustophoretic motion of fluorescent microparticles within the micro-resonators was carefully and accurately measured. The manufactured micro-resonators were validated by comparing the location of the acoustic pressure nodal plane and the average energy density curves derived from numerical and experimental results. Results confirmed that the acoustic radiation force of the induced ultrasonic standing waves drives the microparticles vertically within the micro-resonators and their average energy density increases as the sinusoidal voltage applied to the piezoelectric transducer increases. Semi-empirical correlations were developed for the average energy density, based on experimental results for a wide range of the applied voltage amplitudes. The correlations were in good agreement, within less than 20 % of the experimental values measured for both the half-wavelength and quarter-wavelength micro-resonators.

RESUMEN

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.

RESUMEN

BACKGROUND AIMS: These last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing, including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step for which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. METHODS: An acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using red blood cells suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. RESULTS: With a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a red blood cell/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only two genes, HES4 and MIR-3648-1, were differently expressed compared with the input. CONCLUSIONS: In this study, we developed a continuous cell-washing system based on acoustophoresis. The process allows a theoretically high cell throughput while inducing little gene expression changes. These results indicate that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing.

Asunto(s)

RESUMEN

Although microbubbles are used primarily in the medical industry as ultrasonic contrast agents, they can also be manipulated by acoustic waves for targeted drug delivery, sonothrombolysis and sonoporation. Acoustic waves can also potentially remove microbubbles from tubing systems (e.g., in hemodialysis) to prevent the negative effects associated with circulating microbubbles. A deeper understanding of the interactions between the acoustic radiation force, the microbubble and the channel wall could greatly benefit these applications. In this study, single air-filled microbubbles were injected into a flowing (polydimethylsiloxane) channel and monitored by a high-speed camera while passing through a pulsed ultrasonic wave zone (0.5 MHz). This study compared various bubble sizes, flow rates and acoustic pressure amplitudes to better understand the three physical regimes observed: free bubble translation (away from the wall); on-wall translation; and bubble-wall attachment. Comparison with a theoretical model revealed that the acoustic radiation force needs to exceed the combined repulsive forces (shear lift, wall lubrication and repulsive Van der Waal forces) for the dead state of bubble-wall attachment. The bubble dynamics revealed through this investigation provide an opportunity for efficient positioning of microbubbles in a channel flow, for either in vivo manipulation or removal in biological applications.

Asunto(s)

RESUMEN

A sample preparation step involving dissociation of tissues into their component cells is often required to conduct analysis of nucleic acids and other constituents from tissue samples. Frequently, the extracellular matrix and cell-cell adhesions are disrupted via treatment with a chemical dissociating reagent or various mechanical forces. In this work, a new, high-throughput, multiplexed method of dissociating tissues and cellular aggregates into single cells using ultrasound frequency bath sonication is explored and characterized. Different operating parameters are evaluated, and a treatment protocol with potential for uniform, high-throughput tissue dissociation is compared to the existing best chemical and orbital plate shaking protocol. Metrics such as percent dissociation, cellular recovery, average aggregate size, proportion of various aggregate sizes, membrane circularity, and cellular viability are subsequently assessed and found to be favorable. In optimized conditions, 53 ±â€¯8% of 1 mm biopsy cores are dissociated within 30 min using sonication alone, surpassing leading high-throughput orbital plate shaking techniques five-fold. Chemical digestion is also 2 times more effective when complexed with sonication rather than orbital plate shaking. RNA content, quality, and expression are found to be superior to the standard protocol in terms of transcriptional preservation.

Asunto(s)

RESUMEN

Both passive and active microfluidic chips are used in many biomedical and chemical applications to support fluid mixing, particle manipulations, and signal detection. Passive microfluidic devices are geometry-dependent, and their uses are rather limited. Active microfluidic devices include sensors or detectors that transduce chemical, biological, and physical changes into electrical or optical signals. Also, they are transduction devices that detect biological and chemical changes in biomedical applications, and they are highly versatile microfluidic tools for disease diagnosis and organ modeling. This review provides a comprehensive overview of the significant advances that have been made in the development of microfluidics devices. We will discuss the function of microfluidic devices as micromixers or as sorters of cells and substances (e.g., microfiltration, flow or displacement, and trapping). Microfluidic devices are fabricated using a range of techniques, including molding, etching, three-dimensional printing, and nanofabrication. Their broad utility lies in the detection of diagnostic biomarkers and organ-on-chip approaches that permit disease modeling in cancer, as well as uses in neurological, cardiovascular, hepatic, and pulmonary diseases. Biosensor applications allow for point-of-care testing, using assays based on enzymes, nanozymes, antibodies, or nucleic acids (DNA or RNA). An anticipated development in the field includes the optimization of techniques for the fabrication of microfluidic devices using biocompatible materials. These developments will increase biomedical versatility, reduce diagnostic costs, and accelerate diagnosis time of microfluidics technology.

Asunto(s)

RESUMEN

Supplying a piezoelectric transducer with constant voltage or constant power during a frequency sweep can lead to different results in the determination of the acoustofluidic resonance frequencies, which are observed when studying the acoustophoretic displacements and velocities of particles suspended in a liquid-filled microchannel. In this work, three cases are considered: (1) Constant input voltage into the power amplifier, (2) constant voltage across the piezoelectric transducer, and (3) constant average power dissipation in the transducer. For each case, the measured and the simulated responses are compared, and good agreement is obtained. It is shown that Case 1, the simplest and most frequently used approach, is largely affected by the impedance of the used amplifier and wiring, so it is therefore not suitable for a reproducible characterization of the intrinsic properties of the acoustofluidic device. Case 2 strongly favors resonances at frequencies yielding the lowest impedance of the piezoelectric transducer, so small details in the acoustic response at frequencies far from the transducer resonance can easily be missed. Case 3 provides the most reliable approach, revealing both the resonant frequency, where the power-efficiency is the highest, as well as other secondary resonances across the spectrum.

RESUMEN

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.

Asunto(s)

RESUMEN

In this paper, we proposed an integrated microfluidic device that could demonstrate the non-contact, label-free separation of particles and cells through the combination of inertial microfluidics and acoustophoresis. The proposed device integrated two microfluidic chips which were a PDMS channel chip on top of the silicon-based acoustofluidic chip. The PDMS chip worked by prefocusing the particles/cells through inducing the inertial force of the channel structure. The connected acoustofluidic chips separated particles based on their size through an acoustic radiation force. In the serpentine-shaped PDMS chip, particles formed two lines focusing in the channel, and a trifugal-shaped acoustofluidic chip displaced and separated particles, in which larger particles focused on the central channel and smaller ones moved to the side channels. The simultaneous fluidic works allowed high-efficiency particle separation. Using this novel acoustofluidic device with an inertial microchannel, the separation of particles and cells based on their size was presented and analyzed, and the efficiency of the device was shown. The device demonstrated excellent separation performance with a high recovery ratio (up to 96.3%), separation efficiency (up to 99%), and high volume rate (>100 µL/min). Our results showed that integrated devices could be a viable alternative to current cell separation based on their low cost, reduced sample consumption and high throughput capability.

Asunto(s)

RESUMEN

The analysis of cell motion in an acoustic field is of interest as it can lead to new methods of cell separation, isolation and manipulation for diagnosis and treatment of diseases. Studies of the motion of different species of Leishmania parasites during exposure to ultrasonic standing waves in a microfluidic device allowed identification of acoustic responses of these parasites in their promastigote and amastigote forms. Both forms exhibited a positive acoustic contrast factor and were driven toward the pressure node established in the center of the channel by the acoustically induced radiation force (FR). Promastigotes experience calculated FR amplitudes one order of magnitude larger than those experienced by amastigotes because of the measured differences in volume. The aggregates formed at the pressure node have distinct shapes and stability conditions, for both promastigotes and amastigotes.

Asunto(s)

RESUMEN

Methods for the isolation and analysis of extracellular vesicles (EVs) have been extensively explored in the field of life science and in clinical diagnosis in recent years. The separation and efficient recovery of high-purity target EVs from biological samples are important prerequisites in the study of EVs. So far, commonly used methods of EV separation include ultracentrifugation, filtration, solvent precipitation and immunoaffinity capturing. However, these methods suffer from long processing time, EV damage and low enrichment efficiency. The use of acoustophoretic force facilitates the non-contact label-free manipulation of cells based on their size and compressibility but lacks specificity. Additionally, the acoustophoretic force exerted on sub-micron substances is normally weak and insufficient for separation. Here we present a novel immuno-acoustic sorting technology, where biological substances such as EVs, viruses, and biomolecules, can be specifically captured by antibody/receptor coated microparticles through immunoaffinity, and manipulated by an acoustophoretic force exerted on the microparticles. Using immuno-acoustic sorting technology, we successfully separated and purified HER2-positive EVs for further downstream analysis. This method holds great potential in isolating and purifying specific targets such as disease-related EVs from biological fluids and opens new possibilities for the EV-based early diagnosis and prognosis of diseases.

RESUMEN

In pulp and paper mills, mechanical processes such as screening and washing are commonly used to remove accumulated solid suspensions and concentrate the pulp. For environmental reasons and to optimize paper production, an emerging challenge is to develop alternative methods to concentrate paper pulp between 3 % and 6 % consistency for which the mixed pulp-water flow is complex. Among the proposed solutions in the literature, solutions based on acoustic levitation, also referred as acoustophoresis, of low-consistency pulp have been demonstrated as a potential solution for efficient pulp concentration and water recirculation. However, no sensitivity analysis on the ultrasound and physical parameters was proposed, limiting the extension to a realistic application. Thus, this paper presents a numerical modeling of acoustophoresis for pulp flow concentration in a pipe. For this purpose, the pulp flow is defined as a pseudo-homogenous fluid with a turbulent Low Re k- ∊ formalism, and the pulp particles are considered spherical and deflected by two acoustic forces, namely the acoustic radiation force and the Stokes drag force, both induced by an ultrasound wave generated along the walls of a circular pipe. The combined action of these two forces in the pulp flow enables to concentrate the particles at the center of the pipe. The influences of particle size and mechanical properties, fluid properties and ultrasound parameters are analyzed within a parametric study to optimize the particle deflection and the pulp concentration. The experimental feasibility of the industrial use of acoustophoresis for the concentration of paper pulp is demonstrated with a concentration gain up to 15 %.

RESUMEN

This work illustrates focalization performances of a silicon-based bulk acoustic wave device applied for the separation of specimens owing to micrometric dimensions. Samples are separated in the microfluidic channel by the presence of an acoustic field, which focalizes particles or cells according to their mechanical properties compared to the surrounded medium ones. Design and fabrication processes are reported, followed by focalization performance tests conducted either with synthetic particles or cells. High focalization performances occurred at different microparticle concentrations. In addition, preliminary tests carried out with HL-60 cells highlighted an optimal separation performance at a high flow rate and when cells are mixed with micro and nanoparticles without affecting device focalization capabilities. These encouraging results showed how this bulk acoustic wave device could be exploited to develop a diagnostic tool for early diagnosis or some specific target therapies by separating different kinds of cells or biomarkers possessing different mechanical properties such as shapes, sizes and densities.

RESUMEN

BACKGROUND: Graft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture. RESULTS: PDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft. CONCLUSION: These results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Asunto(s)

RESUMEN

The utilization of bulk acoustic waves from a piezoelectric transducer for selective particle trapping under flow in a microchannel is limited by the high sensitivity of the resonance frequency to tolerances in device geometry, drift during trapping, and variations in the local flow or sample conditions in each channel. This is addressed by detecting the resonance condition based on the impedance minimum obtained by monitoring the amplitude of the stimulation voltage across the piezo transducer and utilizing real-time feedback to control the stimulation frequency. However, this requires an overlap in the frequency bandwidth of the detection and the stimulation system and is also limited by the decline in the acoustic trapping power when the stimulation and resonance frequency measurement are conducted simultaneously. Instead, we present a novel circuit implementation for on-chip real-time resonance frequency measurement and feedback control based on monitoring the current drawn from the amplifier used to stimulate the piezo transducer, since the need for high measurement sensitivity in this mode does not lower the power available for stimulation of the transducer. The enhanced level of control of acoustic trapping utilizing this current mode is validated for various localized channel perturbations, including drift, wash steps, and buffer swaps, as well as for selective sperm cell trapping from a heterogeneous sample that includes lysed epithelial cells.

Asunto(s)

RESUMEN

The use of acoustic waves for microfluidic aggregation has become widespread in chemistry, biology and medicine. Although numerous experimental and analytical studies have been undertaken to study the acoustophoretic aggregation mechanisms, few studies have been conducted to optimise the device design. This paper presents a numerical investigation of the acoustophoresis of microparticles suspended in compressible liquid. The wall of the rectangular microchannel is made of Polydimethylsiloxane (PDMS), and Standing Surface Acoustic Waves (SSAW) are introduced into the channel from the bottom wall. First, the relative amplitude of the acoustic radiation force and the viscous drag force is evaluated for particles of different radii ranging from 0.1µm to 15µm. Only when the particle size is larger than a critical value can the particles accumulate at acoustic pressure nodes (PNs). The efficiency of the particle accumulation depends on the microchannel height, so an extensive parametric study is then undertaken to identify the optimum microchannel height. The optimum height, when normalised by the acoustic wavelength, is found to be between 0.57 and 0.82. These findings provide insights into the design of acoustophoretic devices.

RESUMEN

In cancer research and drug screening, multicellular tumor spheroids (MCTSs) are a popular model to bridge the gap between in vitro and in vivo. However, the current techniques to culture mixed co-culture MCTSs do not mimic the structural architecture and cellular spatial distribution in solid tumors. In this study we present an acoustic trapping-based core-shell MCTSs culture method using sequential seeding of the core and shell cells into microwells coated with a protein repellent coating. Scaffold-free core-shell ovarian cancer OVCAR-8 cell line MCTSs were cultured, stained, cleared and confocally imaged on-chip. Image analysis techniques were used to quantify the shell thickness (23.2 ± 1.8 µm) and shell coverage percentage (91.2 ± 2.8%). We also show that the shell thickness was evenly distributed over the MCTS cores with the exception of being slightly thinner close to the microwell bottom. This scaffold-free core-shell MCTSs formation technique and the analysis tools presented herein could be used as an internal migration assay within the MCTS or to form core-shell MCTS co-cultures to study therapy response or the interaction between tumor and stromal cells.

RESUMEN

This study investigated the application of dual-frequency type dynamic acoustic fields for size-selective particle separation on centimeter scale in a continuous flow. The 3D-printed X-shaped prototype has two inlets and two outlets. The dynamic acoustic field is generated by two transducers positioned under an angle of 60° and operating at slightly different frequencies. The acoustic reflections are eliminated by placing sound-absorbing material inside the prototype and the non-resonant operation is confirmed by the electrical admittance measurements. Numerical calculations suggested that pressure generated by each transducer does not need to have equal amplitude. Computer simulations and lab experiments were carried out for different frequency differences and flow rates. The results demonstrated the ability of dual-frequency dynamic acoustic fields for size-selective particle filtration on centimeter scale, with a total flow rate up to.1Lh-1.

RESUMEN

Culture-expanded mesenchymal stromal cells (MSCs) are promising candidates for clinical cell-based therapies. MSC products are heterogeneous and we therefore investigated whether acoustophoresis, an ultrasound-based separation technology, could be used for the label-free enrichment of functionally different MSC populations. Acoustophoresis uses an ultrasonic standing wave field in a microchannel that differentially affects the movement of cells depending on their acoustophysical properties, such as size, density, and compressibility. Human bone marrow (BM) MSCs were generated by standard adherent culture in xeno-free medium and separated by microchip acoustophoresis. MSCs with up to 20% higher proliferation and 1.7-fold increased clonogenic potential were enriched in the side outlet of the chip compared to the input sample. These cells were significantly smaller (average diameter 14.5 ± 0.4 µm) compared to the center outlet fraction (average diameter 17.1 ± 0.6 µm) and expressed higher levels of genes related to proliferation and stem cell properties (i.e., Ki-67 [1.9-fold], Nanog1 [6.65-fold], Oct4 [2.9-fold], and CXCL12 [1.8-fold], n = 3) in the side outlet compared to input. Fractions of MSCs in G0 /G1 cell cycle phase were significantly enriched in the side fraction and an up to 2.8-fold increase of cells in S/G2 /M phases were observed in center fractions compared to side fractions and 1.3-fold increased compared to the input sample. Acoustophoresis did not compromise MSC phenotype, proliferation, clonogenic capacity, and viability (generally 87-98%), nor did it affect differentiation or immunomodulatory capacities. These results demonstrate that label-free acoustic separation can enrich functionally different MSC subsets which can potentially be employed to produce better-defined stromal cell products from cultured MSCs. Hence, acoustophoresis is a potentially promising separation technology to provide improved cell products for research and possible future clinical use. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Asunto(s)