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.

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.

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.

5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

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.

Hydrogen production techniques based on solar-water splitting have emerged as carbon-free energy systems. Many researchers have developed highly efficient thin-film photoelectrochemical (PEC) devices made of low-cost and earth-abundant materials. However, solar water splitting systems suffer from short lifetimes due to catalyst instability that is attributed to both chemical dissolution and mechanical stress produced by hydrogen bubbles. A recent study found that the nanoporous hydrogel could prevent the structural degradation of the PEC devices. In this study, we investigate the protection mechanism of the hydrogel-based overlayer by engineering its porous structure using the cryogelation technique. Tests for cryogel overlayers with varied pore structures, such as disconnected micropores, interconnected micropores, and surface macropores, reveal that the hydrogen gas trapped in the cryogel protector reduce shear stress at the catalyst surface by providing bubble nucleation sites. The cryogelated overlayer effectively preserves the uniformly distributed platinum catalyst particles on the device surface for over 200 h. Our finding can help establish semi-permanent photoelectrochemical devices to realize a carbon-free society.

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.

We demonstrate the preferential orders of molecular chaperones glucose-regulated protein 94 (GRP94), binding immunoglobulin protein (BiP), and calreticulin (CRT) in an endoplasmic reticulum (ER) fraction from rat liver using columns conjugated with denatured myoglobin, RNase A, or β-lactoglobulin as client proteins in the presence or absence of ATP. The results showed that BiP, CRT, and GRP94 preferentially contributed myoglobin, RNase A, and β-lactoglobulin, respectively, in the presence of ATP. In the absence of ATP, GRP94 and CRT preferentially recognized misfolded myoglobin (α-helix-rich protein), whereas BiP preferentially recognized misfolded RNase A (α-helix/β-sheet mixed protein) and β-lactoglobulin (β-sheet-rich protein). The preferential order of ER chaperones may be dynamically regulated by ER conditions and the higher-order structure of client proteins.

The fabrication of functional tissues is essential for clinical applications such as disease treatment and drug discovery. Recent studies have revealed that the mechanical environments of tissues, determined by geometric cell patterns, material composition, or mechanical properties, play critical roles in ensuring proper tissue function. Here, we propose an acoustophoretic technique using surface acoustic waves to fabricate therapeutic vascular tissue containing a three-dimensional collateral distribution of vessels. Co-aligned human umbilical vein endothelial cells and human adipose stem cells that are arranged in a biodegradable catechol-conjugated hyaluronic acid hydrogel exhibit enhanced cell-cell contacts, gene expression, and secretion of angiogenic and anti-inflammatory paracrine factors. The therapeutic effects of the fabricated vessel constructs are demonstrated in experiments using an ischemia mouse model by exhibiting the remarkable recovery of damaged tissue. Our study can be referenced to fabricate various types of artificial tissues that mimic the original functions as well as structures.

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.