Acoustic Tweezing Research Articles

Acoustic tweezers based on circular, slanted-finger interdigital transducers for dynamic manipulation of micro-objects.

Acoustic tweezing technologies are gaining significant attention from the scientific communities due to their versatility and biocompatibility. This study presents acoustic tweezers based on circular, slanted-finger interdigital transducers (CSFITs), which can steer the propagation direction of surface acoustic waves (SAWs) by tuning the excitation frequency. The CSFITs based acoustic tweezers enable dynamic and reconfigurable manipulation of micro-objects using multi-tone excitation signals. Compared to traditional interdigital transducers that generate and control SAWs along one axis, the CSFITs allow for simultaneously generating and independently controlling SAWs propagating along multiple axes by changing the frequency composition and the phase information in a multi-tone excitation signal. Moreover, the CSFITs based acoustic tweezers can be used for patterning cells/particles in various distributions and translating them along complex paths. We believe that our design is valuable for cellular-scale biological applications, in which on-chip, contactless, biocompatible handling of bioparticles is needed.

Integrin-Targeted Cyclic Forces Accelerate Neural Tube-Like Rosette Formation from Human Embryonic Stem Cells.

Mechanical forces play important roles in human embryonic stem cell (hESC) differentiation. To investigate the impact of dynamic mechanical forces on neural induction of hESCs, this study employs acoustic tweezing cytometry (ATC) to apply cyclic forces/strains to hESCs by actuating integrin-bound microbubbles using ultrasound pulses. Accelerated neural induction of hESCs is demonstrated as the result of combined action of ATC and neural induction medium (NIM). Specifically, application of ATC for 30 min followed by culture in NIM upregulates neuroecdoderm markers Pax6 and Sox1 as early as 6 h after ATC, and induces neural tube-like rosette formation at 48 h after ATC. In contrast, no changes are observed in hESCs cultured in NIM without ATC treatment. In the absence of NIM, ATC application decreases Oct4, but does not increase Pax6 and Sox1 expression, nor does it induce neural rossette formation. The effects of ATC are abolished by inhibition of FAK, myosin activity, and RhoA/ROCK signaling. Taken together, the results reveal a synergistic action of ATC and NIM as an integrated mechanobiology mechanism that requires both integrin-targeted cyclic forces and chemical factors for accelerated neural induction of hESCs.

Acoustic tweezing cytometry for mechanical phenotyping of macrophages and mechanopharmaceutical cytotripsy

Macrophages are immune cells responsible for tissue debridement and fighting infection. Clofazimine, an FDA-approved antibiotic, accumulates and precipitates as rod-shaped, crystal-like drug inclusions within macrophage lysosomes. Drug treatment as well as pathophysiological states could induce changes in macrophage mechanical property which in turn impact their phenotype and function. Here we report the use of acoustic tweezing cytometry as a new approach for in situ mechanical phenotyping of macrophages and for targeted macrophage cytotripsy. Acoustic tweezing cytometry applies ultrasound pulses to exert controlled forces to individual cells via integrin-bound microbubbles, enabling a creep test for measuring cellular mechanical property or inducing irreversible changes to the cells. Our results revealed that macrophages with crystal-like drug inclusions became significantly softer with higher cell compliance, and behaved more elastic with faster creep and recovery time constants. On the contrary, phagocytosis of solid polyethylene microbeads or treatment with soluble clofazimine rendered macrophages stiffer. Most notably, application of ultrasound pulses of longer duration and higher amplitude in ATC actuated the integrin-bound microbubbles to mobilize the crystal-like drug inclusions inside macrophages, turning the rod-shaped drug inclusions into intracellular microblender that effectively destructed the cells. This phenomenon of acoustic mechanopharmaceutical cytotripsy may be exploited for ultrasound activated, macrophage-directed drug release and delivery.

Acoustic levitation and manipulation by a high-frequency focused ring ultrasonic transducer

Recently, acoustic levitation for non-contact micro-particle manipulation has been attracting great interest in physical, biological, and medical applications. Among the state-of-the-art manipulation technologies, single beam acoustic tweezing exhibits advantages of providing stronger trapping force and deeper penetration depth in tissues, inducing less tissue damage, and a simple configuration involving only one device. However, particle trapping by the single beam acoustic tweezer could only be operated on a smooth two-dimensional substrate, which limits the potential for real applications. Here, we report an initial attempt to acoustically levitate an individual micro-particle stably in water and manipulate the levitated micro-particle arbitrarily two-dimensionally by simply employing a 60-MHz focused ring ultrasonic transducer. The proposed working mechanism agrees well with the phenomenon. This approach could not only acoustically levitate and manipulate a micro-particle on a culture dish and on a mylar film, but could also work properly in levitating and manipulating a micro-particle placed inside the polyimide tube. This simple and low-cost approach is extremely useful for effective non-contact micro-particle manipulation without having critical concerns on the substrate properties.

Resonant Acoustic Tweezing Spectroscopy for Small-Volume Noncontact Assessment of Blood Coagulation

Introduction: Measurement and interpretation of mechanical properties of whole blood and blood plasma are important diagnosis and treatment monitoring of various conditions like coagulopathy, hemophilia, sickle cell disease and many cardiovascular disorders. Many of the current techniques like thromboelastography, micro-viscometry or microfluidic devices used for this purpose require a large sample volume and/or may be prone to measurement errors due to sample contact with device walls. To address these issues, we developed a single-drop non-contact method for blood rheological analysis, referred to as “acoustic tweezing rheometry”. With sample volume as small as 4 μL, our innovative technology has been successfully applied for assessment of whole blood and blood plasma coagulation. Here, we present the extension of this technology to resonant spectroscopic measurement of blood viscoelasticity.Materials and Methods: The schematic of the acoustic tweezing device is shown in (Figure 1A). The standing acoustic wave field between the transducer and reflector generates the acoustic radiation force on the biological sample that traps it in a host fluid (e.g., air). Sample tweezing (force-induced deformation and translational motion of the trapped sample) is achieved by amplitude modulation of the acoustic tweezing signal at high frequency and then decrease the frequency continuously until the lower limit for sample trapping is reached. During this frequency sweep, shape changes of the sample were recorded (Figure 1B) by a photodetector and a high-speed camera (Figure 1A). The amplitude-frequency response of the sample was obtained from raw data analysis, with the amplitude being the maximum deflection of the sample height from its equilibrium value. Dynamic (shear) viscosity and elasticity of the sample were assessed from the quality factor of the amplitude-frequency response (Figure 1C) and the resonance frequency, respectively.Results and Discussion: The quality factor analysis predicted that the dynamic viscosity of commercial normal control blood plasma was 1.5 mPa·s at room temperature, which agreed with previous large-sample-volume measurements. Once re-calcified, the resonance frequency of blood plasma and thus its shear elasticity increased due to clot formation until reaching a plateau in 5 min (Figure 1D). Using this graphical output (referred to as “tweezograph”), the following coagulation parameters can be extracted: clot initiation time, clotting rate, clotting time, and maximum clot elasticity.Conclusions: Resonant acoustic tweezing spectroscopy can accurately measure dynamic viscosity and elasticity of whole blood and blood plasma with a small drop of the sample and without artefacts or measurement errors due to sample contact with device walls. This technique can be applied for rapid assessment of whole blood and blood plasma coagulation.Acknowledgments: This study has been supported by U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559 and PCT/US2018/014879. [Display omitted] DisclosuresKhismatullin:Levisonics Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Acoustic Actuation of Integrin-Bound Microbubbles for Mechanical Phenotyping during Differentiation and Morphogenesis of Human Embryonic Stem Cells.

Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, the lack of investigation methods has limited the understanding of how cellular mechanical properties change during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using the recently established in vitro 3D embryogenesis models, ATC measurements reveal that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis.

Acoustic Tweezing Cytometry Induces Rapid Initiation of Human Embryonic Stem Cell Differentiation

Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.

Dynamic measurement of blood viscoelasticity by an oscillatory acoustic tweezing technique

Acoustic tweezing rheometry is an innovative technology for low-volume non-contact rheological analysis of complex fluids characterized by increased sensitivity and accuracy as compared to traditional contact techniques. In this method, a small drop of a fluid sample is levitated in air by acoustic radiation forces and its viscoelasticity at different time instants is measured from drop shape changes. The acoustic tweezing rheometer operates in two different modes: quasi-static and oscillatory. This presentation focuses on the oscillatory technique in which the sample drop is forced into freely decaying shape oscillation by transient modulation of the standing acoustic field. Images of oscillating drops acquired by a high-speed camera are analyzed by a custom MATLAB code to obtain the shape amplitude vs. time curves and then the decay factor and resonance frequency of drop shape oscillation. Dynamic viscoelasticity of the sample (viscosity, relaxation time, and elastic modulus) was measured by applying the experimental data to the analytical formulae, derived from normal mode analysis of drop oscillation for viscoelastic fluid (Maxwell model) and viscoelastic solid (Kelvin-Voigt) materials. Using the oscillatory technique, we measured changes in blood viscoelasticity during coagulation and showed that the Kelvin-Voigt model leads to physically consistent results on rheology of coagulating blood.

Quasi‐static acoustic tweezing thromboelastometry

Background Thromboelastography is widely used as a tool to assess the coagulation status of critical care patients. It allows observation of changes in material properties of whole blood, beginning with early stages of clot formation and ending with clot lysis. However, the contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks. Objectives To develop acoustic tweezing thromboelastometry as a non-contact method for perioperative assessment of blood coagulation. Methods Acoustic tweezing is used to levitate microliter drops of biopolymer and human blood samples. By quasi-statically changing the acoustic pressure we control the sample drop location and deformation. Sample size, deformation and location are determined by digital imaging at each pressure. Results Simple Newtonian liquid solutions maintain a constant, reversible location vs. deformation curve. In contrast, the location/deformation curves for gelatin, alginate, whole blood and blood plasma uniquely change as the samples solidify. Increasing elasticity causes the sample to deform less, leading to steeper stress/strain curves. By extracting a linear regime slope, we show that whole blood or blood plasma exhibits a unique slope profile as it begins to clot. By exposing blood samples to pro- or antithrombotic agents, the slope profile changes, allowing detection of hyper- or hypocoagulable states. Conclusions We demonstrate that quasi-static acoustic tweezing can yield information about clotting onset, maturation and strength. The advantages of small sample size, non-contact and rapid measurement make this technique desirable for real-time monitoring of blood coagulation.

Abstract 520: Acoustic Tweezing Thromboelastometry

Background: Critical care patients such as trauma and major surgery patients often develop coagulopathy due to depletion of both pro- and anti-coagulants. They are at high risk of both bleeding and thrombotic complications and require monitoring of their coagulation status. The contact of a blood sample with artificial surfaces and its exposure to clot activators, which happen in all commercially available coagulation analyzers, may lead to improper assessment of blood coagulation and thus errors in predicting bleeding/thrombosis risks. Objective: Real-time assessment of whole blood or blood plasma coagulation by novel non-contact acoustic tweezing technology. Method: 4-5 microliter drops of whole blood collected from healthy volunteers or commercial control plasma were levitated in air by acoustic radiation forces. Their coagulation kinetics including reaction time, fibrin network formation time (FNFT), clot formation time and maximum clot strength was assessed from mechanical (drop shape) and photo-optical (light intensity) data. FNFT was determined as a difference between mechanical and photo-optical reaction times. Results: Whole blood samples were exposed to pro- or anti-coagulants during levitation in the acoustic tweezing device. Changes in the coagulation status between different experimental groups were detected within 10 minutes. Similarly, less than 7 minutes was required to detect significant changes in reaction time, clot formation time and maximum clot strength between low, normal, and high fibrinogen level control plasma samples. FNFT was shown to be significantly reduced in plasma samples with a higher level of Factor XIII. Conclusions: The acoustic tweezing technology integrates photo-optical tests used in plasma coagulation assays with viscoelastic tests used in whole blood analysis. Its key disruptive features are the increased reliability and accuracy due to non-contact measurement, small sample volume requirement, relatively short procedure time (<10 minutes), and the ability to assess the level of Factor XIII function from FNFT measurements. Our technology addresses a current lack of reliable methods to measure blood coagulation in patients with coagulopathy.

Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation.

Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling functions upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications.

Acoustic Tweezing and Patterning of Concentration Fields in Microfluidics

We demonstrate theoretically that acoustic forces acting on inhomogeneous fluids can be used to pattern and manipulate solute concentration fields into spatio-temporally controllable configurations stabilized against gravity. A theoretical framework describing the dynamics of concentration fields that weakly perturb the fluid density and speed of sound is presented and applied to study manipulation of concentration fields in rectangular-channel acoustic eigenmodes and in Bessel-function acoustic vortices. In the first example, methods to obtain horizontal and vertical multi-layer stratification of the concentration field at the end of a flow-through channel are presented. In the second example, we demonstrate acoustic tweezing and spatio-temporal manipulation of a local high-concentration region in a lower-concentration medium, thereby extending the realm of acoustic tweezing to include concentration fields.

Theoretical Modeling of Biological Fluid Deformation during Dynamic Acoustic Tweezing

Acoustic tweezing rheometry is a novel non-contact method for real-time measurement of rheological properties of biological fluids, without artifacts such as contamination, wall slip effect and other phenomena associated with contact of a sample with device walls. This technique can operate in two regimes - quasi-static elastometry and dynamic measurement of sample viscoelasticity. In this presentation, we report the development of theoretical models for dynamic acoustic tweezing tests in which the sample fluid is modeled using the viscoelastic fluid or viscoelastic solid models (Jeffrey's or Kelvin-Voigt equations, respectively). By asymptotic analysis of the characteristic equation for sample shape oscillation, we obtained analytical relationships between rheological properties (viscosity, relaxation time, elastic modulus) and the frequency and damping rate of shape oscillations, which are directly measured in experiments. To test our models against experiments, we have conducted dynamic acoustic tweezing tests with human whole blood and blood plasma samples. By applying our models to the data of these experiments, we have obtained physically consistent results on blood rheology and showed that the viscosity of blood samples increases by a factor of 4-6 within six minutes of coagulation onset. The presented theoretical models are invaluable for non-contact assessment of biological fluid rheology and understanding the mechanical behavior of biological fluids subjected to the acoustic tweezing field.

Quasi-Static Acoustic Tweezing Thromboelastometry

Acutely unwell patients often develop coagulopathy due to depletion of both pro- and anti-coagulants. They are at high risk of both bleeding and thrombotic complications and require monitoring of their coagulation status. Available contact assays for blood plasma and whole blood coagulation have low predictive power in patients with coagulopathy or take a significant amount of time to obtain diagnostic data. We have developed quasi-static acoustic tweezing thromboelastometry (QATT) as a noncontact method for real-time assessment of blood coagulation. In our method, 4-5 microliter human blood drops are levitated in air by the acoustic radiation force. The sample drop location and deformation are controlled by quasi-statically changing the acoustic pressure. Time-dependent rheological properties of the samples (e.g. sample elasticity) are determined from digital images at each pressure level. Results of our study show that increasing elasticity during coagulation causes blood drops to deform less, leading to steeper deformation/location curves. By extracting a linear regime slope, the samples exhibit a unique slope profile (tweezograph) as they begin to clot. By using tweezographs, we were able to measure the reaction time, coagulation rate and maximum clot stiffness. The exposure of whole blood samples to pro- or anti-coagulants led to significant changes in tweezographs within 10 minutes of sample tweezing, thus allowing detection of hyper- or hypo-coagulable states. Similarly, we detected significant changes between low, normal, and high fibrinogen level control plasma samples, which points out to the ability of our method to measure the fibrinogen level. We demonstrated that QATT can rapidly yield information about coagulation properties of human blood and requires a very small sample volume (∼4 microliter). The advantages of small sample size, non-contact and rapid measurement make this technique desirable for real-time monitoring of blood coagulation in acutely unwell patients.

Estimation of Viscoelastic Properties of Cells Using Acoustic Tweezing Cytometry.

Recently developed acoustic tweezing cytometry uses ultrasound-responsive targeted microbubbles for biomechanical stimulation of live cells at the subcellular level. The purpose of this research was to estimate the viscoelastic characteristics of cells from the displacements of cell-bound microbubbles in response to ultrasound pulses on acoustic tweezing cytometry. Microbubbles were bound to NIH/3T3 fibroblasts and ATDC5 cells through an integrin-cytoskeleton linkage. The evolution of microbubble behaviors under irradiation by ultrasound pulses was captured by a high-speed camera and tracked by a customized algorithm. The total damping constant, stiffness, and rigidity of the cells were estimated by fitting the measured temporal displacement profiles to a Kelvin-Voigt-based model. The mean maximum displacement of the microbubbles attached to NIH/3T3 fibroblasts was much greater than that for ATDC5 cells. The mean fitted damping constant and stiffness ± SD for ATDC5 cells were 28.16 ± 7.08 mg/s and 0.5041 ± 0.1381 mN/m, respectively, and the values for NIH/3T3 fibroblasts were 13.12 ± 4.23 mg/s and 0.2591 ± 0.0715 mN/m. The rigidity for ATDC5 cells was 331.46 ± 106.50 MPa, whereas that for NIH/3T3 fibroblasts was 117.92 ± 34.83 MPa. The Arg-Gly-Asp-integrin-cytoskeleton system of NIH/3T3 fibroblasts appears to be softer than that of ATDC5 cells. The rigidity of ATDC5 cells was significantly greater than that of NIH/3T3 fibroblasts at the 95% confidence level. This strategy provides a novel way to determine the viscoelastic properties of the live cells.

Regulation of Cytoskeleton Contractility and Osteogenesis of Human Mesenchymal Stem Cells using Acoustic Tweezing Cytometry (ATC)

Human mesenchymal stem cells (hMSCs) have great potential for cellular therapeutics for treating degenerative bone diseases such as osteoporosis. However, in vitro cell culture conditions for hMSC osteogenesis still remain suboptimal. In this study, we used an ultrasound-based technique, acoustic tweezing cytometry (ATC), to apply dynamic subcellular mechanical forces to enhance in vitro hMSC osteogenesis. In previous work, we have shown that single ATC application for 10-20 sec could lead to a sustained increase of the total cytoskeleton contractility of hMSCs for 30 min. As the contractile force of hMSCs has been implicated as a functional predictor of their long-term osteogenesis, we studied the effect of ATC stimulation on hMSC contractility for 24 hr. To identify the optimal experimental conditions to promote hMSC osteogenesis, we explored the effects of different ATC doses, as well as cell shape and substrate rigidity on cytoskeleton contractility of hMSCs. Substrate rigidity was modulated by varying the height of an elastomeric micropost array. We used micro-contact printing to print different adhesive islands to modulate cell shape. Moreover, we characterized the effect of ATC to regulate osteogenic differentiation of hMSCs. We plan to study how the RhoA/ROCK/myosin signaling axis is involved in intracellular force transduction by ATC in hMSCs in the future. In summary, our results showed that ATC was capable of modulating cytoskeleton contractility and enhancing osteogenesis of hMSCs in vitro for cell-therapeutics applications for treating bone diseases.

Acoustic Tweezing Cytometry (ATC) on Dissociated Human Embryonic Stem Cells (HESCS)

Human embryonic stem cells (hESCs) have wide potential use in regenerative medicine and cell therapies. However, large-scale cell expansion is limited as hESCs undergo differentiation and apoptosis after single-cell dissociation. Currently, Rho-associated kinase (ROCK) inhibitors are used in hESC culture to maintain cell survival, yet with the risk of long-term negative effects on hESCs. In previous work, we have shown acoustic tweezing cytometry (ATC), an ultrasound-based subcellular mechanical stimulation technique, can significantly improve survival rate of dissociated hESCs while maintaining their pluripotency without the presence of ROCK inhibitor Y-27632. Herein, we further investigated the impact of ATC on dissociated hESCs and the mechanisms underlying the enhancement of hESC survival by ATC. To determine optimal ATC treatment conditions, we exposed hESCs to ATC treatments with different acoustic pressure levels (i.e., 0.05 - 0.25 MPa) and exposure frequencies, and assayed their effects on hESC survival. To explore the role of E-cadherin and integrin mediated signaling pathways in hESC survival, we used microbubbles targeted to E-cadherin and integrin on the cells in ATC application, and evaluated the effects on hESCs in terms of survival rate, cell area, colony size, and stemness. In addition, we monitored cell intracellular cytoskeleton contractility using a fluorescence-labeled micropost system in the initial cell spreading stage, and compared the contractility changes over time under various ATC stimulation conditions. In summary, results from this study will help us to develop an effective treatment scheme using ATC to enhance dissociated hESC survival and reveal the insights about mechanotransductive mechanisms involving E-cadherin and integrin-mediated adhesion signaling for hESC survival and pluripotency maintenance.

Improving Survival of Disassociated Human Embryonic Stem Cells by Mechanical Stimulation Using Acoustic Tweezing Cytometry

Dissociation-induced apoptosis of human embryonic stem cells (hESCs) hampers their large-scale culture. Herein we leveraged the mechanosensitivity of hESCs and employed, to our knowledge, a novel technique, acoustic tweezing cytometry (ATC), for subcellular mechanical stimulation of disassociated single hESCs to improve their survival. By acoustically actuating integrin-bound microbubbles (MBs) to live cells, ATC increased the survival rate and cloning efficiency of hESCs by threefold. A positive correlation was observed between the increased hESC survival rate and total accumulative displacement of integrin-anchored MBs during ATC stimulation. ATC may serve as a promising biocompatible tool to improve hESC culture.

Two-Bubble Acoustic Tweezing Cytometry for Biomechanical Probing and Stimulation of Cells

The study of mechanotransduction relies on tools that are capable of applying mechanical forces to elicit and assess cellular responses. Here we report a new (to our knowledge) technique, called two-bubble acoustic tweezing cytometry (TB-ATC), for generating spatiotemporally controlled subcellular mechanical forces on live cells by acoustic actuation of paired microbubbles targeted to the cell adhesion receptor integrin. By measuring the ultrasound-induced activities of cell-bound microbubbles and the actin cytoskeleton contractile force responses, we determine that TB-ATC elicits mechanoresponsive cellular changes via cyclic, paired displacements of integrin-bound microbubbles driven by the attractive secondary acoustic radiation force (sARF) between the bubbles in an ultrasound field. We demonstrate the feasibility of dual-mode TB-ATC for both subcellular probing and mechanical stimulation. By exploiting the robust and unique interaction of ultrasound with microbubbles, TB-ATC provides distinct advantages for experimentation and quantification of applied forces and cellular responses for biomechanical probing and stimulation of cells.

Activation of a Bacterial Mechanosensitive Channel in Mammalian Cells by Cytoskeletal Stress

Cells can sense a myriad of mechanical stimuli. Mechanosensitive channel of large conductance (MscL) found in bacteria is a well-characterized mechanosensitive channel that rapidly responds to an increase in turgor pressure. Functional expression of MscL in mammalian cells has recently been demonstrated, revealing that molecular delivery or transport can be achieved by charge-induced activation of MscL. Despite a well-accepted mechanism for MscL activation by membrane tension in bacteria, it is not clear whether and how MscL can be opened by other modes of force transduction in mammalian cells. In this work, we used a variety of techniques to characterize the gating of MscL expressed in mammalian cells, using both wild type and a G22S mutant which activates at a lower threshold. In particular, employing a new technique, acoustic tweezing cytometry (ATC), we show that ultrasound actuation of integrin-bound microbubbles can lead to MscL opening and that ATC induced MscL activation was dependent on the functional linkage of the microbubbles with an intact actin cytoskeleton. Our results indicate that localized mechanical stress can mediate opening of MscL that requires force transduction through the actin cytoskeleton, revealing a new mode of MscL activation that may prove to be a useful tool for mechanobiology and drug delivery research.