Maturation of Human iPSC-Derived Cardiac Microfiber with Electrical Stimulation Device.

Here an electrical stimulation system is described for maturing microfiber-shaped cardiac tissue (cardiac microfibers, CMFs). The system enables stable culturing of CMFs with electrical stimulation by placing the tissue between electrodes. The electrical stimulation device provides an electric field covering whole CMFs within the stimulation area and can control the beating of the cardiac microfibers. In addition, CMFs under electrical stimulation with different frequencies are examined to evaluate the maturation levels by their sarcomere lengths, electrophysiological characteristics, and gene expression. Sarcomere elongation (14% increase compared to control) is observed at day 10, and a significant upregulation of electrodynamic properties such as gap junction protein alpha 1 (GJA1) and potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) (maximum fourfold increase compared to control) is observed at day 30. These results suggest that electrically stimulated cultures can accelerate the maturation of microfiber-shaped cardiac tissues compared to those without electrical stimulation. This model will contribute to the pathological research of unexplained cardiac diseases and pharmacologic testing by stably constructing matured CMFs.

Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer.

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

Spatiotemporal single-cell tracking analysis in 3D tissues to reveal heterogeneous cellular response to mechanical stimuli.

Mechanical stimuli have been recognized as important for tissue maturation, homeostasis and constructing engineered three-dimensional (3D) tissues. However, we know little about the cellular mechanical response in tissues that could be considerably heterogeneous and spatiotemporally dynamic due to the complex structure of tissues. Here, we report a spatiotemporal single-cell tracking analysis of in vitro 3D tissues under mechanical stretch, to reveal the heterogeneous cellular behavior by using a developed stretch and optical live imaging system. The system could affect the cellular orientation and directly measure the distance of cells in in vitro 3D myoblast tissues (3DMTs) at the single-cell level. Moreover, we observed the spatiotemporal heterogeneous cellular locomotion and shape changes under mechanical stretch in 3DMTs. This single-cell tracking analysis can become a principal method to investigate the heterogeneous cellular response in tissues and provide insights that conventional analyses have not yet offered.

Hydrogel-Sheathed hiPSC-Derived Heart Microtissue Enables Anchor-Free Contractile Force Measurement.

In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force-sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long-term culture. This paper proposes a hydrogel-sheathed human induced pluripotent stem cell (hiPSC)-derived heart microtissue (H3 M) that can provide an anchor-free contractile force measurement platform in commonly used multi-well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3 M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT.

Mechanical effect of reconstructed shapes of autologous ossicles on middle ear acoustic transmission.

Conductive hearing loss is caused by a variety of defects, such as chronic otitis media, osteosclerosis, and malformation of the ossicles. In such cases, the defective bones of the middle ear are often surgically reconstructed using artificial ossicles to increase the hearing ability. However, in some cases, the surgical procedure does not result in increased hearing, especially in a difficult case, for example, when only the footplate of the stapes remains and all of the other bones are destroyed. Herein, the appropriate shapes of the reconstructed autologous ossicles, which are suitable for various types of middle-ear defects, can be determined by adopting an updating calculation based on a method that combines numerical prediction of the vibroacoustic transmission and optimization. In this study, the vibroacoustic transmission characteristics were calculated for bone models of the human middle ear by using the finite element method (FEM), after which Bayesian optimization (BO) was applied. The effect of the shape of artificial autologous ossicles on the acoustic transmission characteristics of the middle ear was investigated with the combined FEM and BO method. The results suggested that the volume of the artificial autologous ossicles especially has a great influence on the numerically obtained hearing levels.

Schwann cell-encapsulated chitosan-collagen hydrogel nerve conduit promotes peripheral nerve regeneration in rodent sciatic nerve defect models.

Chitosan has various tissue regeneration effects. This study was designed to investigate the nerve regeneration effect of Schwann cell (SC)-encapsulated chitosan-collagen hydrogel nerve conduit (CCN) transplanted into a rat model of sciatic nerve defect. We prepared a CCN consisting of an outer layer of chitosan hydrogel and an inner layer of collagen hydrogel to encapsulate the intended cells. Rats with a 10-mm sciatic nerve defect were treated with SCs encapsulated in CCN (CCN+), CCN without SCs (CCN-), SC-encapsulated silicone tube (silicone+), and autologous nerve transplanting (auto). Behavioral and histological analyses indicated that motor functional recovery, axonal regrowth, and myelination of the CCN+ group were superior to those of the CCN- and silicone+ groups. Meanwhile, the CCN- and silicone+ groups showed no significant differences in the recovery of motor function and nerve histological restoration. In conclusion, SC-encapsulated CCN has a synergistic effect on peripheral nerve regeneration, especially axonal regrowth and remyelination of host SCs. In the early phase after transplantation, SC-encapsulated CCNs have a positive effect on recovery. Therefore, using SC-encapsulated CCNs may be a promising approach for massive peripheral nerve defects.

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound.

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l-lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.

A Novel Rat Model of Embolic Cerebral Ischemia Using a Cell-Implantable Radiopaque Hydrogel Microfiber.

The failure of neuroprotective treatment-related clinical trials, including stem cell therapies, may be partially due to a lack of suitable animal models. We have developed a stem cell-implantable radiopaque hydrogel microfiber that can survive for a long time in vivo. The microfiber is made of barium alginate hydrogel containing zirconium dioxide, fabricated in a dual coaxial laminar flow microfluidic device. We aimed to develop a novel focal stroke model using this microfiber. Using male Sprague-Dawley rats (n=14), a catheter (inner diameter, 0.42 mm; outer diameter, 0.55 mm) was navigated from the caudal ventral artery to the left internal carotid artery using digital subtraction angiography. A radiopaque hydrogel microfiber (diameter, 0.4 mm; length, 1 mm) was advanced through the catheter by slow injection of heparinized physiological saline to establish local occlusion. Both 9.4-T magnetic resonance imaging at 3 and 6 h and 2% 2,3,5-triphenyl tetrazolium chloride staining at 24 h after stroke model creation were performed. Neurological deficit score and body temperature were measured. The anterior cerebral artery-middle cerebral artery bifurcation was selectively embolized in all rats. Median operating time was 4 min (interquartile range [IQR], 3-8 min). Mean infarct volume was 388 mm3 (IQR, 354-420 mm3) at 24 h after occlusion. No infarction of the thalamus or hypothalamus was seen. Body temperature did not change significantly over time (P = 0.204). However, neurological deficit scores before and at 3, 6, and 24 h after model creation differed significantly (P < 0.001). We present a novel rat model of focal infarct restricted to the middle cerebral artery territory using a radiopaque hydrogel microfiber positioned under fluoroscopic guidance. By comparing the use of stem cell-containing versus non-containing fibers in this stroke model, it would be possible to determine the efficacy of "pure" cell transplantation in treating stroke.

Acoustic-responsive carbon dioxide-loaded liposomes for efficient drug release.

The role of liposomes as drug carriers has been investigated. Ultrasound-based drug release methods have been developed for on-demand drug delivery. However, the acoustic responses of current liposome carriers result in low drug release efficiency. In this study, CO2-loaded liposomes were synthesized under high pressure from supercritical CO2 and irradiated with ultrasound at 237 kHz to demonstrate their superior acoustic responsiveness. When liposomes containing fluorescent drug models were irradiated with ultrasound under acoustic pressure conditions that are safe for the human body, CO2-loaded liposomes synthesized using supercritical CO2 had 17.1 times higher release efficiency than liposomes synthesized using the conventional Bangham method. In particular, the release efficiency of CO2-loaded liposomes synthesized using supercritical CO2 and monoethanolamine was 19.8 times higher than liposomes synthesized using the conventional Bangham method. These findings on the release efficiency of acoustic-responsive liposomes suggest an alternative liposome synthesis strategy for on-demand release of drugs by ultrasound irradiation in future therapies.

Near-Infrared-Triggered On-Demand Controlled Release of Adeno-Associated Virus from Alginate Hydrogel Microbeads with Heat Transducer for Gene Therapy.

Gene therapy using adeno-associated virus (AAV) has potential as a radical treatment modality for genetic diseases such as sensorineural deafness. To establish clinical applications, it is necessary to avoid immune response to AAV by controlled release system of AAV. Here, a near-infrared (NIR)-triggered on-demand AAV release system using alginate hydrogel microbeads with a heat transducer is proposed. By using a centrifuge-based microdroplet shooting device, the microbeads encapsulating AAV with Fe3 O4 microparticles (Fe3 O4 -MPs) as a heat transducer are fabricated. Fe3 O4 -MPs generated heat by NIR enhanced the diffusion speed of the AAV, resulting in the AAV being released from the microbeads. By irradiating the microbeads encapsulating fluorescent polystyrene nanoparticles (FP-NPs) (viral model) with NIR, the fluorescence intensity decreased only for FP-NPs with a diameter of 20 nm and not for 100 or 200 nm, confirming that this system can release virus with a diameter of several tens of nanometers. By irradiating NIR to the AAV-encapsulating microbeads with Fe3 O4 -MPs, the AAV is released on demand, and gene transfection to cells by AAV is confirmed without loss of viral activity. The NIR-triggered AAV release system proposed in this study increases the number of alternatives for the method of drug release in gene therapy.

Combined analysis of finite element model and audiometry provides insights into the pathogenesis of conductive hearing loss.

The middle ear transmits sound to the inner ear via vibrations in the eardrum and ossicles, and damage to the middle ear results in conductive hearing loss. Although conductive hearing loss can be corrected by surgery, the currently available clinical investigations cannot always diagnose the ossicular chain pathology underlying the conductive hearing loss, and even intraoperative findings can be equivocal. Acoustic analysis using finite element models (FEMs) can simulate the sound pressure change at an arbitrary site for each frequency. FEMs are used in acoustic engineering to simulate the frequency-dependent sound pressure distribution at discrete cells in a simulated model and analyze the effects of specific parameters on the audiogram. However, few reports have compared the numerical results obtained using FEMs with data from clinical cases. We used FEMs to simulate audiograms of the air-bone gap (ABG) for various ossicular chain defects and compared these with preoperative audiograms obtained from 44 patients with a normal tympanic membrane who had otosclerosis, middle ear malformations or traumatic ossicular disruption. The simulated audiograms for otosclerosis and attic fixation of the malleus-incus complex both exhibited an up-slope but could be distinguished from each other based on the ABG at 1000 Hz. The simulated audiogram for incudostapedial joint discontinuity exhibited a peak at around 750 Hz and a down-slope above 1000 Hz. In general, the simulated audiograms for otosclerosis, attic fixation and incudostapedial joint discontinuity were consistent with those obtained from clinical cases. Additional simulations indicated that changes in ossicular mass had relatively small effects on ABG. Furthermore, analyses of combination pathologies suggested that the effects of one defect on ABG were added to those of the other defect. These FEM-based findings provide insights into the pathogenesis of conductive hearing loss due to otosclerosis, middle ear malformations and traumatic injury.

Effect of the area of a lithium niobate transducer on the efficiency of ultrasonic atomization driven by resonance vibration.

In recent years, individual control of one's personal environment has been drawing increasing attention due to the growing interest in health care. Wearable devices are especially useful because of their controllability regardless of location. Humidity is one of the inevitable factors in the personal environment as a preventive against infectious diseases. Although atomization devices are commonly used as a method of humidity control, at present, there are no wearable humidity control devices. Vibration of a lithium niobate (LN) device in the thickness mode is a promising piezoelectric method for miniaturization of atomization devices for humidity control. To miniaturize the atomization device, the transducer size needs to be small not so much as to decrease the atomization efficiency. However, the effect of the device area on the atomization efficiency of LN at a size suitable for mounting in wearable devices has not been studied. Here, we conducted an atomization demonstration of LN devices with different sizes to evaluate particle size and atomization efficiency. Furthermore, to reveal the relationship between vibration behavior and atomization efficiency, resonance vibration in the MHz frequency band was evaluated by the finite element method and an impedance analyzer. The results showed that the peak size of water particles atomized by each device was in the range of 3.2 to 4.2 µm, which is smaller than particles produced by typical piezoelectric ceramics. Moreover, the best LN size for efficient atomization was found to be 8 mm × 10 mm among the five LN device sizes used in experiments. From the relationship between vibration behavior and atomization efficiency, the size of the transducer was suggested to affect the vibration mode. The obtained result suggested that the LN device is suitable for small wearable nebulizer devices.

Quantitative Analysis of Acoustic Pressure for Sonophoresis and Its Effect on Transdermal Penetration.

Ultrasound facilitates the penetration of macromolecular compounds through the skin and offers a promising non-invasive technique for transdermal delivery. However, technical difficulties in quantifying ultrasound-related parameters have restricted further analysis of the sonophoresis mechanism. In this study, we devise a bolt-clamped Langevin transducer-based sonophoresis device that enables us to measure with a thin lead zirconate titanate (PZT) sensor. One-dimensional acoustic theory accounting for wave interaction at the skin interface indicates that the acoustic pressure and cavitation onset on the skin during sonophoresis are sensitive to the subcutaneous support, meaning that there is a strong need to perform the pressure measurement in an experimental environment replacing the human body. From a series of the experiments with our new device, the transdermal penetration of polystyrene, silica and gold nanoparticles is found to depend on the size and material of the particles, as well as the hardness of the subcutaneous support material. We speculate from the acoustic pressure measurement that the particles' penetration results from the mechanical action of cavitation.

A novel fabrication process of up-scalable microfiber-shaped tendon-like tissue with high cell density for uniformed macroscale assembly.

This paper describes up-scalable microfiber-shaped tissues for macroscale tendon tissue reconstruction in vitro. C3H10T1/2 cells were encapsulated in a calcium alginate hydrogel microfiber that was fabricated via a double coaxial microfluidic device. The C3H10T1/2 cells gradually merged to construct the microfiber-shaped tendon-like tissue. Our microfiber-shaped tendon-like tissues were alive and maintained their microfiber-shaped morphology over 600 days. Immunostaining and real-time quantitative polymerase chain reaction analyses showed that our fabricated microfiber-shaped tendon-like tissue properly expressed tenomodulin and the orientation of the filaments of actin, which are one of the characteristics of tendon tissue in vivo. Furthermore, a macroscale tendon tissue assembly with ∼1 cm in length and ∼200 µm in thickness was successfully constructed by bundling the microfiber-shaped tendon-like tissues together. This feature enabled us to fabricate a macroscale tendon tissue with uniform cell distribution. We believe that our fabricated microfiber-shaped tendon-like tissue would be a suitable strategy to reconstruct tendon tissue in vitro for the treatments of tendon-related injuries.

Janus Hydrogel Microbeads for Glucose Sensing with pH Calibration.

We present fluorescent Janus hydrogel microbeads for continuous glucose sensing with pH calibration. The Janus hydrogel microbeads, that consist of fluorescent glucose and pH sensors, were fabricated with a UV-assisted centrifugal microfluidic device. The microbead can calibrate the pH values of its surroundings and enables accurate measurements of glucose within various pH conditions. As a proof of concept, we succeeded in obtaining the accurate value of glucose concentration in a body-fluid-like sample solution. We believe that our fluorescent microbeads, with pH calibration capability, could be applied to fully implantable sensors for continuous glucose monitoring.

Focused surface acoustic wave locally removes cells from culture surface.

Regenerative medicine and drug development require large numbers of high-quality cells, usually delivered from in vitro culturing. During culturing, the appearance of unwanted cells and an inability to remove them without damaging or losing most if not all the surrounding cells in the culture reduce the overall quality of the cultured cells. This is a key problem in cell culturing, as is the inability to sample cells from a culture as desired to verify the quality of the culture. Here, we report a method to locally remove cells from an adherent cell culture using a 100.4 MHz focused surface acoustic wave (SAW) device. After exposing a plated C2C12 mouse myoblast cell culture to phosphate buffered solution (PBS), ultrasound from the SAW device transmitted into the cell culture via a coupling water droplet serves to detach a small grouping of cells. The cells are removed from an area 6 × 10-3 mm2, equivalent to about 12 cells, using a SAW device-Petri dish water gap of 1.5 mm, a PBS immersion time of 300 s, and an input voltage of 75 V to the SAW device. Cells were released as desired 90% of the time, releasing the cells from the target area nine times out of ten runs. In the one trial in ten that fails, the cells partially release and remain attached due to inter-cellular binding. By making it possible to target and remove small groups of cells as desired, the quality of cell culturing may be significantly improved. The small group of cells may be considered a colony of iPS cells. This targeted cell removal method may facilitate sustainable, contamination-free, and automated refinement of cultured cells.

Suspension culture in a T-flask with acoustic flow induced by ultrasonic irradiation.

Suspension culture is an essential large-scale cell culture technique for biopharmaceutical development and regenerative medicine. To transition from monolayer culture on the culture surface of a flask to suspension culture in a bioreactor, a pre-specified cell number must first be reached. During this period of preparation for suspension culture, static suspension culture in a flask is generally performed because the medium volume is not large enough to use a paddle to circulate the medium. However, drawbacks to this static method include cell sedimentation, leading to high cell density near the bottom and resulting in oxygen and nutrient deficiencies. Here, we propose a suspension culture method with acoustic streaming induced by ultrasonic waves in a T-flask to create a more homogeneous distribution of oxygen, nutrients, and waste products during the preparation period preceding large-scale suspension culture in a bioreactor. To demonstrate the performance of the ultrasonic method, Chinese hamster ovary cells were cultured for 72 h. Results showed that, on average, the cell proliferation was improved by 40% compared with the static method. Thus, the culture time required to achieve a 1000-fold increase could be reduced by 32 h (a 14% reduction) compared with the static method. Furthermore, the ultrasonic irradiation did not compromise the metabolic activity of the cells cultured using the ultrasonic method. These results demonstrate the effectiveness of the ultrasonic method for accelerating the transition to large-scale suspension culture.

Detachment of RAW264.7 macrophages from a culture dish using ultrasound excited by a Langevin transducer.

To study the relationship between macrophages and antigens, an efficient culture method for macrophages is important. During culture, macrophages adhering to the culture surface are difficult to harvest by general trypsinization. Thus, prolonged trypsinization or cell scraping has been used to detach macrophages. However, prolonged trypsinization has a negative effect on cell viability, and the detachment efficiency with cell scrapers depends highly on the skill of a technician. Therefore, we developed a macrophage-detaching method by combining trypsin-EDTA and ultrasonic vibration to detach cells from a ubiquitous culture vessel. We fabricated a device that propagated ultrasound to a φ-35-mm culture dish from underneath. To demonstrate our concept, RAW264.7 cells were used as model cells and exposed to several detaching conditions to evaluate the effects of our developed method. In addition to the proposed method, as traditional detaching methods, simple trypsinization with trypsin-EDTA and manual cell scraping were performed. Furthermore, to determine the optimal intensity of the ultrasound, input voltages into the ultrasound transducer of 200, 225, and 250 V were used. As a result, the number of live cells detached by the developed method with an input amplitude of 225 V was approximately 4.8 times more than that by simple trypsinization and approximately 4.3 times more than that by scraping. Furthermore, the proliferation and phagocytosis level of the cells were increased by the developed method at 225 V, while no significant difference was found in metabolism. Thus, the developed method improves culture efficiency and cell functions without causing metabolic disorders.

Cell-encapsulated chitosan-collagen hydrogel hybrid nerve guidance conduit for peripheral nerve regeneration.

Nerve guidance conduits (NGCs) composed of biocompatible polymers have been attracting attention as an alternative for autograft surgery in peripheral nerve regeneration. However, the nerve tissues repaired by NGCs often tend to be inadequate and lead to functional failure because of the lack of cellular supports. This paper presents a chitosan-collagen hydrogel conduit containing cells to induce peripheral nerve regeneration with cellular support. The conduit composed of two coaxial hydrogel layers of chitosan and collagen is simply made by molding and mechanical anchoring attachment with holes made on the hydrogel tube. A chitosan layer strengthens the conduit mechanically, and a collagen layer provides a scaffold for cells supporting the axonal extension. The conduits of different diameters (outer diameter approximately 2-4 mm) are fabricated. The conduit is bioabsorbable with lysozyme, and biocompatible even under bio absorption. In the neuron culture demonstration, the conduit containing Schwann cells induced the extension of the axon of neurons directed to the conduit. Our easily fabricated conduit could help the high-quality regeneration of peripheral nerves and contribute to the nerve repair surgery.