Dynamic reorganization of multivesicular bodies and exosome production impacted by sonoporation.

Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.

Brown Adipose Tissue as a Unique Niche for Islet Organoid Transplantation: Insights From In Vivo Imaging.

Background: Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery.

Methods: A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase.

Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force.

Multichannel resonant acoustic rheometry system for quantification of coagulation of multiple human plasma samples.

Resonant Acoustic Rheometry (RAR), a newly developed ultrasound-based technique for non-contact characterization of soft viscoelastic materials, has shown promise for quantitative viscoelastic assessment of temporally changing soft biomaterials in real time, and may be used to monitor blood coagulation process. Here, we report the development of a novel, multichannel RAR (mRAR) system for simultaneous measurements of multiple temporally evolving samples and demonstration of its use for monitoring the coagulation of multiple small-volume plasma samples. The mRAR system was constructed using an array of 4 custom-designed ultrasound transducers at 5.

Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry.

Acoustic tweezing cytometry (ATC) is an ultrasound-based biophysical technique that has shown the capability to promote differentiation of human pluripotent stem cells (hPSCs). This study systematically examined how hPSCs respond to cyclic mechanical strains applied by ATC via displacement of integrin-bound microbubbles (averaged diameter of 4.3 µm) using ultrasound pulses (acoustic pressure 0.

Crossover of surface waves and capillary-viscous-elastic transition in soft biomaterials detected by resonant acoustic rheometry.

Viscoelastic properties of hydrogels are important for their application in science and industry. However, rheological assessment of soft hydrogel biomaterials is challenging due to their complex, rapid, and often time-dependent behaviors. Resonant acoustic rheometry (RAR) is a newly developed technique capable of inducing and measuring resonant surface waves in samples in a non-contact fashion.

Multichannel Resonant Acoustic Rheometry System for Rapid and Efficient Quantification of Human Plasma Coagulation.

Resonant Acoustic Rheometry (RAR), a newly developed ultrasound-based technique for non-contact characterization of soft viscoelastic materials, has shown promise for quantitative assessment of plasma coagulation by monitoring the entire dynamic process in real time. Here, we report the development of a multichannel RAR (mRAR) system for simultaneous monitoring of the coagulation of multiple small-volume plasma samples, a capability that is critical to efficiently provide improved assessment of coagulation. The mRAR system was constructed using an array of 4 custom-designed ultrasound transducers at 5.

Resonant Acoustic Rheometry to Measure Coagulation Kinetics in Hemophilia A and Healthy Plasma: A Novel Viscoelastic Method.

Compared with conventional coagulation tests and factor-specific assays, viscoelastic hemostatic assays (VHAs) can provide a more thorough evaluation of clot formation and lysis but have several limitations including clot deformation. In this proof-of-concept study, we test a noncontact technique, termed resonant acoustic rheometry (RAR), for measuring the kinetics of human plasma coagulation. Specifically, RAR utilizes a dual-mode ultrasound technique to induce and detect surface oscillation of blood samples without direct physical contact and measures the resonant frequency of the surface oscillation over time, which is reflective of the viscoelasticity of the sample.

Resonant acoustic rheometry for non-contact characterization of viscoelastic biomaterials.

Resonant Acoustic Rheometry (RAR) is a new, non-contact technique to characterize the mechanical properties of soft and viscoelastic biomaterials, such as hydrogels, that are used to mimic the extracellular matrix in tissue engineering. RAR uses a focused ultrasound pulse to generate a microscale perturbation at the sample surface and tracks the ensuing surface wave using pulse-echo ultrasound. The frequency spectrum of the resonant surface waves is analyzed to extract viscoelastic material properties.

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation.

Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies.

Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo.

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds.

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).

High Frequency Spectral Ultrasound Imaging to Detect Metastasis in Implanted Biomaterial Scaffolds.

For most cancers, metastasis is the point at which disease is no longer curable. Earlier detection of metastasis, when it is undetectable by current clinical methods, may enable better outcomes. We have developed a biomaterial implant that recruits metastatic cancer cells in mouse models of breast cancer.

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.

Rapid translocation of pluripotency-related transcription factors by external uniaxial forces.

Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors.

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.

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).

Multimode ultrasound viscoelastography for three-dimensional interrogation of microscale mechanical properties in heterogeneous biomaterials.

Both static and time-dependent mechanical factors can have a profound impact on cell and tissue function, but it is challenging to measure the mechanical properties of soft materials at the scale which cells sense. Multimode ultrasound viscoelastography (MUVE) uses focused ultrasound pulses to both generate and image deformations within soft hydrogels non-invasively, at sub-millimeter resolution, and in 3D. The deformation and strain over time data are used to extract quantitative parameters that describe both the elastic and viscoelastic properties of the material.

High-frequency spectral ultrasound imaging (SUSI) visualizes early post-traumatic heterotopic ossification (HO) in a mouse model.

Purpose: Early treatment of heterotopic ossification (HO) is currently limited by delayed diagnosis due to limited visualization at early time points. In this study, we validate the use of spectral ultrasound imaging (SUSI) in an animal model to detect HO as early as one week after burn tenotomy.

Methods: Concurrent SUSI, micro CT, and histology at 1, 2, 4, and 9weeks post-injury were used to follow the progression of HO after an Achilles tenotomy and 30% total body surface area burn (n=3-5 limbs per time point).

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.

Microscale characterization of the viscoelastic properties of hydrogel biomaterials using dual-mode ultrasound elastography.

Characterization of the microscale mechanical properties of biomaterials is a key challenge in the field of mechanobiology. Dual-mode ultrasound elastography (DUE) uses high frequency focused ultrasound to induce compression in a sample, combined with interleaved ultrasound imaging to measure the resulting deformation. This technique can be used to non-invasively perform creep testing on hydrogel biomaterials to characterize their viscoelastic properties.

High resolution Physio-chemical Tissue Analysis: Towards Non-invasive In Vivo Biopsy.

Conventional gold standard histopathologic diagnosis requires information of both high resolution structural and chemical changes in tissue. Providing optical information at ultrasonic resolution, photoacoustic (PA) technique could provide highly sensitive and highly accurate tissue characterization noninvasively in the authentic in vivo environment, offering a replacement for histopathology. A two-dimensional (2D) physio-chemical spectrogram (PCS) combining micrometer to centimeter morphology and chemical composition simultaneously can be generated for each biological sample with PA measurements at multiple optical wavelengths.

Ultrasound Imaging Techniques for Spatiotemporal Characterization of Composition, Microstructure, and Mechanical Properties in Tissue Engineering.

Ultrasound techniques are increasingly being used to quantitatively characterize both native and engineered tissues. This review provides an overview and selected examples of the main techniques used in these applications. Grayscale imaging has been used to characterize extracellular matrix deposition, and quantitative ultrasound imaging based on the integrated backscatter coefficient has been applied to estimating cell concentrations and matrix morphology in tissue engineering.

Calibration and Evaluation of Ultrasound Thermography Using Infrared Imaging.

Real-time monitoring of the spatiotemporal evolution of tissue temperature is important to ensure safe and effective treatment in thermal therapies including hyperthermia and thermal ablation. Ultrasound thermography has been proposed as a non-invasive technique for temperature measurement, and accurate calibration of the temperature-dependent ultrasound signal changes against temperature is required. Here we report a method that uses infrared thermography for calibration and validation of ultrasound thermography.