Quantifying cell adhesion through forces generated by acoustic streaming.

The strength of cell adhesion is important in understanding the cell's health and in culturing them. Quantitative measurement of cell adhesion strength is a significant challenge in bioengineering research. For this, the present study describes a system that can measure cell adhesion strength using acoustic streaming induced by Lamb waves. Cells are cultured on an ultrasound transducer using a range of preculture and incubation times with phosphate-buffered saline (PBS) just before the measurement. Acoustic streaming is then induced using several Lamb wave intensities, exposing the cells to shear flows and eventually detaching them. By relying upon a median detachment rate of 50 %, the corresponding detachment force, or force of cell adhesion, was determined to be on the order of several nN, consistent with previous reports. The stronger the induced shear flow, the more cells were detached. Further, we employed a preculture time of 8 to 24 h and a PBS incubation time of 0 to 60 min, producing cell adhesion forces that varied from 1.2 to 13 nN. Hence, the developed system can quantify cell adhesion strength over a wide range, possibly offering a fundamental tool for cell-based bioengineering.

Nonlinear Tactile Estimation Model Based on Perceptibility of Mechanoreceptors Improves Quantitative Tactile Sensing.

Tactile sensing has attracted significant attention as a tactile quantitative evaluation method because the tactile sensation is an important factor while evaluating consumer products. Although the human tactile perception mechanism has nonlinearity, previous studies have often developed linear regression models. In contrast, this study proposes a nonlinear tactile estimation model that can estimate sensory evaluation scores from physical measurements. We extracted features from the vibration data obtained by a tactile sensor based on the perceptibility of mechanoreceptors. In parallel, a sensory evaluation test was conducted using 10 evaluation words. Then, the relationship between the extracted features and the tactile evaluation results was modeled using linear/nonlinear regressions. The best model was concluded by comparing the mean squared error between the model predictions and the actual values. The results imply that there are multiple evaluation words suitable for adopting nonlinear regression models, and the average error was 43.8% smaller than that of building only linear regression models.

Well-free agglomeration and on-demand three-dimensional cell cluster formation using guided surface acoustic waves through a couplant layer.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

A Model for Estimating Tactile Sensation by Machine Learning Based on Vibration Information Obtained while Touching an Object.

The tactile sensation is an important indicator of the added value of a product, and it is thus important to be able to evaluate this sensation quantitatively. Sensory evaluation is generally used to quantitatively evaluate the tactile sensation of an object. However, statistical evaluation of the tactile sensation requires many participants and is, thus, time-consuming and costly. Therefore, tactile sensing technology, as opposed to sensory evaluation, is attracting attention. In establishing tactile sensing technology, it is necessary to estimate the tactile sensation of an object from information obtained by a tactile sensor. In this research, we developed a tactile sensor made of two-layer silicone rubber with two strain gauges in each layer and obtained vibration information as the sensor traced an object. We then extracted features from the vibration information using deep autoencoders, following the nature of feature extraction by neural firing due to vibrations perceived within human fingers. We also conducted sensory evaluation to obtain tactile scores for different words from participants. We finally developed a tactile sensation estimation model for each of the seven samples and evaluated the accuracy of estimating the tactile sensation of unknown samples. We demonstrated that the developed model can properly estimate the tactile sensation for at least four of the seven samples.

Development of accurate temperature regulation culture system with metallic culture vessel demonstrates different thermal cytotoxicity in cancer and normal cells.

Hyperthermia has been studied as a noninvasive cancer treatment. Cancer cells show stronger thermal cytotoxicity than normal cells, which is exploited in hyperthermia. However, the absence of methods evaluating the thermal cytotoxicity in cells prevents the development of hyperthermia. To investigate the thermal cytotoxicity, culture temperature should be regulated. We, thus, developed a culture system regulating culture temperature immediately and accurately by employing metallic culture vessels. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used for models of cancer and normal cells. The findings showed cancer cells showed stronger thermal cytotoxicity than normal cells, which is quantitatively different from previous reports. This difference might be due to regulated culture temperature. The thermal stimulus condition (43 °C/30 min) was, further, focused for assays. The mRNA expression involving apoptosis changed dramatically in cancer cells, indicating the strong apoptotic trend. In contrast, the mRNA expression of heat shock protein (HSP) of normal cells upon the thermal stimulus was stronger than cancer cells. Furthermore, exclusively in normal cells, HSP localization to nucleus was confirmed. These movement of HSP confer thermotolerance to cells, which is consistent with the different thermal cytotoxicity between cancer and normal cells. In summary, our developed system can be used to develop hyperthermia treatment.

Propagating acoustic waves on a culture substrate regulate the directional collective cell migration.

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.

Acoustic streaming induced by MHz-frequency ultrasound extends the volume limit of cell suspension culture.

Large-scale cell suspension culture technology opens up opportunities for numerous medical and bioengineering applications. For these purposes, scale-up of the culture system is paramount. For initial small-scale culture, a simple static suspension culture (SSC) is generally employed. However, cell sedimentation due to the lack of agitation limits the culture volume feasible for SSC. Thus, when scaling up, cell suspensions must be manually transferred from the culture flask to another vessel suitable for agitation, which increases the risk of contamination and human error. Ideally, the number of culture transfer steps should be kept to a minimum. The present study describes the fabrication of an ultrasonic suspension culture system that stirs cell suspensions with the use of acoustic streaming generated by ultrasound irradiation at a MHz frequency. This system was applied to 100-mL suspension cultures of Chinese hamster ovary cells-a volume ten-fold larger than that generally used. The cell proliferation rate in this system was 1.88/day when applying an input voltage of 40 V to the ultrasonic transducer, while that of the SSC was 1.14/day. Hence, the proposed method can extend the volume limit of static cell suspension cultures, thereby reducing the number of cell culture transfer steps.

Travelling ultrasound promotes vasculogenesis of three-dimensional-monocultured human umbilical vein endothelial cells.

To generate three-dimensional tissue in vitro, promoting vasculogenesis in cell aggregates is an important factor. Here, we found that ultrasound promoted vasculogenesis of human umbilical vein endothelial cells (HUVECs). Promotion of HUVEC network formation and lumen formation were observed using our method. In addition to morphological evaluations, protein expression was quantified by western blot assays. As a result, expression of proteins related to vasculogenesis and the response to mechanical stress on cells was enhanced by exposure to ultrasound. Although several previous studies have shown that ultrasound may promote vasculogenesis, the effect of ultrasound was unclear because of unregulated ultrasound, the complex culture environment, or two-dimensional-cultured HUVECs that cannot form a lumen structure. In this study, regulated ultrasound was propagated on three-dimensional-monocultured HUVECs, which clarified the effect of ultrasound on vasculogenesis. We believe this finding may be an innovation in the tissue engineering field.

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.

Containerless Bioorganic Reactions in a Floating Droplet by Levitation Technique Using an Ultrasonic Wave.

To ensure sustainable consumption and production patterns, alternative process design without plastics for chemical and biological reactions will benefit future generations. Reaction flasks used in chemical and biological laboratories have been made from glass, metals, and plastics so far. If containerless processing can be realized, researchers will have a next-generation reaction process, which will be reactor and plastic-free, and without risks of unforeseen issues induced by contact with reactions flasks, including contamination and alteration of the reactants. Here, polymerization, click chemistry, and enzymatic reactions can proceed effectively in a floating droplet at a node of standing wave generated by ultrasonic levitation. These results demonstrate that floating droplets levitated by acoustic waves can represent a revolutionary containerless reactor for performing various reactions in the fields of chemistry and biology.

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.

Collective cell migration of fibroblasts is affected by horizontal vibration of the cell culture dish.

Regulating the collective migration of cells is an important issue in bioengineering. Enhancing or suppressing cell migration and controlling the migration direction is useful for various physiological phenomena such as wound healing. Several methods of migration regulation based on different mechanical stimuli have been reported. While vibrational stimuli, such as sound waves, show promise for regulating migration, the effect of the vibration direction on collective cell migration has not been studied in depth. Therefore, we fabricated a vibrating system that can apply horizontal vibration to a cell culture dish. Here, we evaluated the effect of the vibration direction on the collective migration of fibroblasts in a wound model comprising two culture areas separated by a gap. Results showed that the vibration direction affects the cell migration distance: vibration orthogonal to the gap enhances the collective cell migration distance while vibration parallel to the gap suppresses it. Results also showed that conditions leading to enhanced migration distance were also associated with elevated glucose consumption. Furthermore, under conditions promoting cell migration, the cell nuclei become elongated and oriented orthogonal to the gap. In contrast, under conditions that reduce the migration distance, cell nuclei were oriented to the direction parallel to the gap.

Low-frequency mechanical vibration induces apoptosis of A431 epidermoid carcinoma cells.

Cancer research is increasingly focused on discovering strategies to induce cancer cell apoptosis without affecting surrounding normal cells. One potential biocompatible method is mechanical vibration, which has been developed as part of the emerging field of mechanomedicine. Previous studies of mechanical vibration have employed high-frequency vibration, which damages healthy cells. In this study, we examined the effects of brief (1 h) low-frequency (20 Hz) mechanical vibration on glucose consumption and survival (apoptosis, necrosis, HMGB1 release) of the human epidermoid carcinoma cell line A431. We found that apoptosis, but not necrosis, was significantly increased at 48 h after mechanical vibration compared with cells maintained in static culture. In keeping with this, extracellular release of HMGB1, a necrosis marker, was lower in cultures of A431 cells subjected to mechanical vibration compared with control cells. Glucose consumption was increased in the first 24 h after mechanical vibration but returned to control levels before the onset of apoptosis. Although the precise intracellular mechanisms by which low-frequency mechanical vibration triggers apoptosis of A431 cells is unknown, these results suggest a possible role for metabolic pathways. Mechanical vibration may thus represent a novel application of mechanomedicine to cancer therapy.

Homogenization of initial cell distribution by secondary flow of medium improves cell culture efficiency.

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.

Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration.

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations.

Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting RVs that have 1-3 antinodes with ultra-low-density enzyme spread on each substrate of SCPs, 87.8% of hiPSCs were successfully detached from a 5-layer SCP compared to 30.8% detached by the conventional enzymatic method. hiPSC viability was similar after either method. Moreover, hiPSCs detached by the RV method maintained their undifferentiated state. Additionally, hiPSCs after long-term culture (10 passages) kept excellent detachment efficiency, had the normal karyotypes, and maintained the undifferentiated state and pluripotency. These results indicated that the RV method has definite advantages over the conventional enzymatic method in the scalable culture of hiPSCs using SCPs.

Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Formation of Large Scaffold-Free 3-D Aggregates in a Cell Culture Dish by Ultrasound Standing Wave Trapping.

Cellular aggregates that mimic cell-cell interactions in vitro are essential for biological research. This study introduces a method to form large scaffold-free 3-D aggregates in a clinically ubiquitous cell culture dish using kilohertz-order ultrasound standing wave trapping (USWT). We fabricated an aggregate formation system in which a 60-mm dish was set above a Langevin transducer via water. The transducer was excited at 110.8 kHz, and then C2C12 myoblasts were injected into the dish and trapped at the node position of the standing wave. The diameter and thickness of the formed aggregate were 8 and 2.7 mm, respectively, which are larger than those of aggregates formed previously by USWT. Moreover, we confirmed that >94% of cells constituting the aggregates survived 9 h, and the protein expression of cells was not altered significantly. This method can be applied to form aggregates with high functionality, which contributes to the development of biological research methodology.

Method of localized removal of cells using a bolt-clamped Langevin transducer with an ultrasonic horn.

Cell isolation by eliminating undesirable cell aggregations or colonies with low activity is essential to improve cell culture efficiency. Moreover, when creating tissues from induced pluripotent stem cells, residual undifferentiated cells must be removed to prevent tumor formation in vivo. Here, we evaluated the use of ultrasonic irradiation, which can apply energy locally without contact, and proposed a method to eliminate cells in a small area of culture by ultrasonic irradiation from a Langevin transducer. We constructed a device that incorporated a bolt-clamped 19.84 kHz Langevin transducer with an ultrasonic horn and determined the optimal conditions for stable elimination of cells in small areas of a 35-mm culture dish. The optimal conditions were as follows: number of cycles = 400, clearance distance = 1 mm, volume of medium = 4 mL, and distance from the center of culture surface = 0 mm. The mean cell elimination area under these conditions was 0.097 mm2. We also evaluated the viability of neighboring cells after ultrasonic irradiation by fluorescent staining and found that most cells around the elimination area survived. These findings suggest that the proposed method has potential for localized elimination of cells without the need for contact with the cell surface.