A Novel Acoustic Modulation of Oscillating Thin Elastic Membrane for Enhanced Streaming in Microfluidics and Nanoscale Liposome Production.

Liposomes are widely utilized in therapeutic nanosystems as promising drug carriers for cancer treatment, which requires a meticulous synthesis approach to control the nanoprecipitation process. Acoustofluidic platforms offer a favorable synthesis environment by providing robust agitation and rapid mixing. Here, a novel high-throughput acoustofluidic micromixer is presented for a solvent and solvent-free synthesis of ultra-small and size-tunable liposomes. The size-tunability is achieved by incorporating glycerol as a new technique into the synthesis reagents, serving as a size regulator. The proposed device utilizes the synergistic effects of vibrating trapped microbubbles and an oscillating thin elastic membrane to generate vigorous acoustic microstreaming. The working principle and mixing mechanism of the device are explored numerically and experimentally. The platform exhibits remarkable mixing efficacy for aqueous and viscous solutions at flow rates up to 8000 µL/h, which makes it unique for high-throughput liposome formation and preventing aggregation. As a proof of concept, this study investigates the impact of phospholipid type and concentration, flow rate, and glycerol on the size and size distribution of liposomes. The results reveal a significant size reduction, from ≈900 nm to 40 nm, achieved by merely introducing 75% glycerol into the synthesis reagents, highlighting an innovative approach toward size-tunable liposomes.

Biomodulatory Effects of Molecular Delivery in Human T Cells Using 3D-Printed Acoustofluidic Devices.

OBJECTIVE: Cell-based therapies have shown significant promise for treating many diseases, including cancer. Current cell therapy manufacturing processes primarily utilize viral transduction to insert genomic material into cells, which has limitations, including variable transduction efficiency and extended processing times. Non-viral transfection techniques are also limited by high variability or reduced molecular delivery efficiency. Novel 3D-printed acoustofluidic devices are in development to address these challenges by delivering biomolecules into cells within seconds via sonoporation. METHODS: In this study, we assessed biological parameters that influence the ultrasound-mediated delivery of fluorescent molecules (i.e., calcein and 150 kDa FITC-Dextran) to human T cells using flow cytometry and confocal imaging. RESULTS: Low cell plating densities (100,000 cells/mL) enhanced molecular delivery compared to higher cell plating densities (p < 0.001), even though cells were resuspended at equal concentrations for acoustofluidic processing. Additionally, cells in the S phase of the cell cycle had enhanced intracellular delivery compared to cells in the G2/M phase (p < 0.001) and G0/G1 phase (p < 0.01), while also maintaining higher viability compared to G0/G1 phase (p < 0.001). Furthermore, the calcium chelator (EGTA) decreased overall molecular delivery levels. Confocal imaging indicated that the actin cytoskeleton had important implications on plasma membrane recovery dynamics after sonoporation. In addition, confocal imaging indicates that acoustofluidic treatment can permeabilize the nuclear membrane, which could enable rapid intranuclear delivery of nucleic acids. CONCLUSIONS: The results of this study demonstrate that a 3D-printed acoustofluidic device can enhance molecular delivery to human T cells, which may enable improved techniques for non-viral processing of cell therapies.

Surface Acoustic Wave-Enhanced Multi-View Acoustofluidic Rotation Cytometry (MARC) for Pre-Cytopathological Screening.

Cytopathology, crucial in disease diagnosis, commonly uses microscopic slides to scrutinize cellular abnormalities. However, processing high volumes of samples often results in numerous negative diagnoses, consuming significant time and resources in healthcare. To address this challenge, a surface acoustic wave-enhanced multi-view acoustofluidic rotation cytometry (MARC) technique is developed for pre-cytopathological screening. MARC enhances cellular morphology analysis through comprehensive and multi-angle observations and amplifies subtle cell differences, particularly in the nuclear-to-cytoplasmic ratio, across various cell types and between cancerous and normal tissue cells. By prioritizing MARC-screened positive cases, this approach can potentially streamline traditional cytopathology, reducing the workload and resources spent on negative diagnoses. This significant advancement enhances overall diagnostic efficiency, offering a transformative vision for cytopathological screening.

Acoustic levitation as a tool for cell-driven self-organization of human cell spheroids during long-term 3D culture.

Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix.

Microfluidic Acoustic Method for High Yield Extraction of Cell-Free DNA in Low-Volume Plasma Samples.

Cell-free DNA has many applications in clinical medicine, in particular in cancer diagnosis and cancer treatment monitoring. Microfluidic-based solutions could provide solutions for rapid, cheaper, decentralized detection of cell-free tumoral DNA from a simple blood draw, or liquid biopsies, replacing invasive procedures or expensive scans. In this method, we present a simple microfluidic system for the extraction of cell-free DNA from low volume of plasma samples (≤500 µL). The technique is suitable for either static or continuous flow systems and can be used as a stand-alone module or integrated within a lab-on-chip system. The system relies on a simple yet highly versatile bubble-based micromixer module whose custom components can be fabricated with a combination of low-cost rapid prototyping techniques or ordered via widely available 3D-printing services. This system is capable of performing cell-free DNA extractions from small volumes of blood plasma with up to a tenfold increase in capture efficiency when compared to control methods.

An enhanced tilted-angle acoustic tweezer for mechanical phenotyping of cancer cells.

Acoustofluidic devices becomes one of the emerging and versatile tools for many biomedical applications. Most of the previous acoustofluidic devices are used for cells manipulation, and the few devices for cell phenotyping with a limitation in throughput. In this study, an enhanced tilted-angle (ETA) acoustofluidic device is developed and applied for mechanophenotyping of live cells. The ETA Device consists of an interdigital transducer which is positioned along a microfluidic channel. An inclination angle of 5° is introduced between the interdigital transducer and the liquid flow direction. The pressure nodes formed inside the acoustofluidic field in the channel deflect the biological cells from their original course in accordance with their mechanical properties, including volume, compressibility, and density. The threshold power for fully converging the cells to the pressure node is used to calculate the acoustic contrast factor. To demonstrate the ETA device in cell mechanophenotyping, and distinguishing between different cell types, further experimentation is carried out by using A549 (lung cancer cells), MDB-MA-231 (breast cancer cells), and leukocytes. The resulting acoustic contrast factors for the lung and breast cancer cells are different from that of the leukocytes by 27.9% and 21.5%, respectively. These results suggest this methodology can successfully distinguish and phenotype different cell types based on the acoustic contrast factor.

An ultra-compact acoustofluidic device based on the narrow-path travelling surface acoustic wave (np-TSAW) for label-free isolation of living circulating tumor cells.

Obtaining highly purified intact living cells from complex environments has been a challenge, such as the isolation of circulating tumor cells (CTCs) from blood. In this work, we demonstrated an acoustic-based ultra-compact device for cell sorting, with a chip size of less than 2 × 1.5 cm2. This single actuator device allows non-invasive and label-free isolation of living cells, offering greater flexibility and applicability. The device performance was optimized with different-sized polystyrene (PS) particles and blood cells spiked with cancer cells. Using the narrow-path travelling surface acoustic wave (np-TSAW), precise isolation of 10 µm particles from a complex mixture of particles (5, 10, 20 µm) and separation of 8 µm and 10 µm particles was achieved. The purified collection of 10 µm particles with high separation efficiency (98.75%) and high purity (98.1%) was achieved by optimizing the input voltage. Further, we investigated the isolation and purification of CTCs (MCF-7, human breast cancer cells) from blood cells with isolation efficiency exceeding 98% and purity reaching 93%. Viabilities of the CTCs harvested from target-outlet were all higher than 97% after culturing for 24, 48, and 72 h, showing good proliferation ability. This novel ultra-miniaturized microfluidic chip demonstrates the ability to sorting cells with high-purity and label-free, providing an attractive miniaturized system alternative to traditional sorting methods.

Acoustofluidic assembly of primary tumor-derived organotypic cell clusters for rapid evaluation of cancer immunotherapy.

Cancer immunotherapy shows promising potential for treating breast cancer. While patients may have heterogeneous treatment responses for adjuvant therapy, it is challenging to predict an individual patient's response to cancer immunotherapy. Here, we report primary tumor-derived organotypic cell clusters (POCCs) for rapid and reliable evaluation of cancer immunotherapy. By using a label-free, contactless, and highly biocompatible acoustofluidic method, hundreds of cell clusters could be assembled from patient primary breast tumor dissociation within 2 min. Through the incorporation of time-lapse living cell imaging, the POCCs could faithfully recapitulate the cancer-immune interaction dynamics as well as their response to checkpoint inhibitors. Superior to current tumor organoids that usually take more than two weeks to develop, the POCCs can be established and used for evaluation of cancer immunotherapy within 12 h. The POCCs can preserve the cell components from the primary tumor due to the short culture time. Moreover, the POCCs can be assembled with uniform fabricate size and cell composition and served as an open platform for manipulating cell composition and ratio under controlled treatment conditions with a short turnaround time. Thus, we provide a new method to identify potentially immunogenic breast tumors and test immunotherapy, promoting personalized cancer therapy.

Assessment of silicon, glass, FR4, PDMS and PMMA as a chip material for acoustic particle/cell manipulation in microfluidics.

In the present study, the capabilities of different chip materials for acoustic particle manipulation have been assessed with the same microfluidic device architecture, under the same actuator and flow conditions. Silicon, glass, epoxy with fiberglass filling (FR4), polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA) are considered as chip materials. The acoustophoretic chips in this study were manufactured with four different fabrication methods: plasma etching, chemical etching, micromachining and molding. A novel chip material, FR4, has been employed as a microfluidic chip material in acoustophoretic particle manipulation for the first time in literature, which combines the ease of manufacturing of polymer materials with improved acoustic performance. The acoustic particle manipulation performance is evaluated through acoustophoretic focusing experiments with 2µm and 12µm polystyrene microspheres and cultured breast cancer cell line (MDA-MB-231). Unlike the common approach in the literature, the piezoelectric materials were actuated with partitioned cross-polarized electrodes which allowed effective actuation of different family of chip materials. Different from previous studies, this study evaluates the performance of each acoustophoretic device through the perspective of synchronization of electrical, vibrational and acoustical resonances, considers the thermal performance of the chip materials with their effects on cell viability as well as manufacturability and scalability of their fabrication methods. We believe our study is an essential work towards the commercialization of acoustophoretic devices since it brings a critical understanding of the effect of chip material on device performance as well as the cost of achieving that performance.

On the application of sound radiation force for focusing of powder stream in directed energy deposition.

One of the challenges in directed energy deposition via powder feeding (DED-PF) is the powder stream divergence that results in low catchment efficiency (i.e., the fraction of particles added to the melt pool). This article introduces a new ultrasound-based powder focusing method referred to as ultrasound particle lensing (UPL), tailored for powder used in DED-PF. The method uses an ultrasound phased array to produce a small volume of high-intensity ultrasound with the required period averaged sound intensity profile. UPL was used to acoustically focus streams of Ti64 and SS 316L particles with an average size of 89µm and a particle speed of 0.6 m/s, exiting from a DED-PF nozzle analog. The e-1 powder stream widths downstream of the resulting force fields for both materials were reduced by 30%. The experimental results closely match Lagrangian and Eulerian simulations of the process. This novel setup offers the possibility of fast control of the powder stream divergence angle and effective diameter in the process zone during the DED-PF process. This will in turn improve the feature resolution and catchment efficiency of the process.

A review of acoustofluidic separation of bioparticles.

Acoustofluidics is an emerging interdisciplinary research field that involves the integration of acoustics and microfluidics to address challenges in various scientific areas. This technology has proven to be a powerful tool for separating biological targets from complex fluids due to its label-free, biocompatible, and contact-free nature. Considering a careful designing process and tuning the acoustic field particles can be separated with high yield. Recently the advancement of acoustofluidics led to the development of point-of-care devices for separations of micro particles which address many of the limitations of conventional separation tools. This review article discusses the working principles and different approaches of acoustofluidic separation and provides a synopsis of its traditional and emerging applications, including the theory and mechanism of acoustofluidic separation, blood component separation, cell washing, fluorescence-activated cell sorting, circulating tumor cell isolation, and exosome isolation. The technology offers great potential for solving clinical problems and advancing scientific research.

Rapid Profiling of Tumor-Immune Interaction Using Acoustically Assembled Patient-Derived Cell Clusters.

Tumor microenvironment crosstalk, in particular interactions between cancer cells, T cells, and myeloid-derived suppressor cells (MDSCs), mediates tumor initiation, progression, and response to treatment. However, current patient-derived models such as tumor organoids and 2D cultures lack some essential niche cell types (e.g., MDSCs) and fail to model complex tumor-immune interactions. Here, the authors present the novel acoustically assembled patient-derived cell clusters (APCCs) that can preserve original tumor/immune cell compositions, model their interactions in 3D microenvironments, and test the treatment responses of primary tumors in a rapid, scalable, and user-friendly manner. By incorporating a large array of 3D acoustic trappings within the extracellular matrix, hundreds of APCCs can be assembled within a petri dish within 2 min. Moreover, the APCCs can preserve sensitive and short-lived (≈1 to 2-day lifespan in vivo) tumor-induced MDSCs and model their dynamic suppression of T cell tumor toxicity for up to 24 h. Finally, using the APCCs, the authors succesully model the combinational therapeutic effect of a multi-kinase inhibitor targeting MDSCs (cabozantinib) and an anti-PD-1 immune checkpoint inhibitor (pembrolizumab). The novel APCCs may hold promising potential in predicting treatment response for personalized cancer adjuvant therapy as well as screening novel cancer immunotherapy and combinational therapy.

Sonoporation: Past, Present, and Future.

A surge of research in intracellular delivery technologies is underway with the increased innovations in cell-based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high-throughput delivery that is critical for providing the desired cell quantity for cell-based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and has been demonstrated for delivering a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble-based sonoporation methods usually require special contrast agents. Bubble-based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non-bubble-based sonoporation mechanisms are under development. This review will cover both the bubble-based and non-bubble-based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.

Soft-Contact Acoustic Microgripper Based on a Controllable Gas-Liquid Interface for Biomicromanipulations.

The manipulation of microscale bioentities is desired in many biological and biomedical applications. However, the potential unobservable damage to bioparticles due to rigid contact has always been a source of concern. Herein, a soft-contact acoustic microgripper to handle microparticles to improve the interaction safety is introduced. The system takes advantage of the acoustic-enhanced adhesion of flexible gas-liquid interfaces to capture-release, transport, and rotate the target, such as microbeads (20-65 µm) and zebrafish embryos (from 950 µm to 1.4 mm). The gas-liquid interface generated at the tip of a microcapillary can be precisely controlled by a pneumatic pressure source. The gas-liquid interface oscillation excited by acoustic energy imposes coupled radiation force and drag force on the microparticles, enabling multidimensional movements. Experiments with the microbeads are conducted to evaluate the claimed function and quantify the key parameters that influence the manipulation result. Additionally, 250 zebrafish embryos are captured, transported, and rotated. The hatching rate of the 250 manipulated embryos is approximately 98% similar to that of the nonmanipulated group, which proves the noninvasiveness of the method. The derived theories and experimental data indicate that the developed soft-contact microgripper is functional and beneficial for biological and medical applications.

Acoustofluidic Droplet Sorter Based on Single Phase Focused Transducers.

Droplet microfluidics has revolutionized the biomedical and drug development fields by allowing for independent microenvironments to conduct drug screening at the single cell level. However, current microfluidic sorting devices suffer from drawbacks such as high voltage requirements (e.g., >200 Vpp), low biocompatibility, and/or low throughput. In this article, a single-phase focused transducer (SPFT)-based acoustofluidic chip is introduced, which outperforms many microfluidic droplet sorting devices through high energy transmission efficiency, high accuracy, and high biocompatibility. The SPFT-based sorter can be driven with an input power lower than 20 Vpp and maintain a postsorting cell viability of 93.5%. The SPFT sorter can achieve a throughput over 1000 events per second and a sorting purity up to 99.2%. The SPFT sorter is utilized here for the screening of doxorubicin cytotoxicity on cancer and noncancer cells, proving its drug screening capability. Overall, the SPFT droplet sorting device shows great potential for fast, precise, and biocompatible drug screening.

Rapid Formation of Multicellular Spheroids in Boundary-Driven Acoustic Microstreams.

3D cell spheroid culture has emerged as a more faithful recreation of cell growth environment compared to conventional 2D culture, as it can maintain tissue structures, physicochemical characteristics, and cell phenotypes. The majority of current spheroid formation methods are limited to a physical agglomeration of the desired cell type, and then relying on cell capacity to secrete extracellular matrix to form coherent spheroids. Hence, apart from being time-consuming, their success in leading to functional spheroid formation is also cell-type dependent. In this study, a boundary-driven acoustic microstreaming tool is presented that can simultaneously congregate cells and generate sturdy cell clusters through incorporating a bioadhesive such as collagen for rapid production of spheroids. The optimized mixture of type I collagen (0.42 mg mL-1 ) and methylcellulose (0.4% w/v ) accelerates the coagulation of cell-matrix as fast as 10 s while avoiding their adhesion to the device, and thereby offering easy spheroid retrieval. The versatility of the platform is shown for the production of MDA-MB-231 and MCF-7 spheroids, multicellular spheroids, and composite spheroids made of cells and microparticles. The ability to produce densely packed spheroids embedded within a biomimetic extracellular matrix component, along with rapid formation and easy collection of spheroids render the proposed device a step in technology development required to realize potentials of 3D constructs such as building blocks for the emerging field of bottom-up tissue engineering.

Flexible/Bendable Acoustofluidics Based on Thin-Film Surface Acoustic Waves on Thin Aluminum Sheets.

In this paper, we explore the acoustofluidic performance of zinc oxide (ZnO) thin-film surface acoustic wave (SAW) devices fabricated on flexible and bendable thin aluminum (Al) foils/sheets with thicknesses from 50 to 1500 µm. Directional transport of fluids along these flexible/bendable surfaces offers potential applications for the next generation of microfluidic systems, wearable biosensors and soft robotic control. Theoretical calculations indicate that bending under strain levels up to 3000 µÎµ causes a small frequency shift and amplitude change (<0.3%) without degrading the acoustofluidic performance. Through systematic investigation of the effects of the Al sheet thickness on the microfluidic actuation performance for the bent devices, we identify the optimum thickness range to both maintain efficient microfluidic actuation and enable significant deformation of the substrate, providing a guide to design such devices. Finally, we demonstrate efficient liquid transportation across a wide range of substrate geometries including inclined, curved, vertical, inverted, and lateral positioned surfaces using a 200 µm thick Al sheet SAW device.

Comparison of Acoustic Streaming Flow Patterns Induced by Solid, Liquid and Gas Obstructions.

In this study, acoustic streaming flows inside micro-channels induced by three different types of obstruction-gaseous bubble, liquid droplet and solid bulge-are compared and investigated experimentally by particle tracking velocimetry (PTV) and numerically using the finite element method (FEM). The micro-channels are made by poly(dimethylsiloxane) (PDMS) using soft lithography with low-cost micro-machined mold. The characteristic dimensions of the media are 0.2 mm in diameter, and the oscillation generated by piezoelectric actuators has frequency of 12 kHz and input voltages of 40 V. The experimental results show that in all three obstruction types, a pair of counter-rotating vortical patterns were observed around the semi-circular obstructions. The gaseous bubble creates the strongest vortical streaming flow, which can reach a maximum of 21 mm/s, and the largest u component happens at Y/D = 0. The solid case is the weakest of the three, which can only reach 2 mm/s. The liquid droplet has the largest v components and speed at Y/D = 0.5 and Y/D = 0.6. Because of the higher density and incompressibility of liquid droplet compared to the gaseous bubble, the liquid droplet obstruction transfers the oscillation of the piezo plate most efficiently, and the induced streaming flow region and average speed are both the largest of the three. An investigation using numerical simulation shows that the differing interfacial conditions between the varying types of obstruction boundaries to the fluid may be the key factor to these differences. These results suggest that it might be more energy-efficient to design an acoustofluidic device using a liquid droplet obstruction to induce the stronger streaming flow.

The acoustic radiation force of a focused ultrasound beam on a suspended eukaryotic cell.

Although ultrasound tools for manipulating and permeabilizing suspended cells have been available for nearly a century, accurate prediction of the distribution of acoustic radiation force (ARF) continues to be a challenge. We therefore developed an analytical model of the acoustic radiation force (ARF) generated by a focused Gaussian ultrasound beam incident on a eukaryotic cell immersed in an ideal fluid. The model had three layers corresponding to the nucleus, cytoplasm, and membrane, of a eukaryotic cell. We derived an exact expression for the ARF in relation to the geometrical and acoustic parameters of the model cell components. The mechanics of the cell membrane and nucleus, the relative width of the Gaussian beam, the size, position and aspect ratio of the cell had significant influence on the ARF. The model provides a theoretical basis for improved acoustic control of cell trapping, cell sorting, cell assembly, and drug delivery.

Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.