伊藤 弘明

This paper focuses on the comparison of red blood cell (RBC) deformability under continuous and repetitive loadings. We utilized a feedback position-control system and a narrow microfluidic channel for applying different deformation patterns on RBCs. According to the analyses of shape recoveries with different patterns, we found, for the first time, that the mechanical responses of RBCs upon continuous and repetitive loadings are almost the same within the error among cellular individualities as long as the total duration of the loading is the same. The result indicates that the internal mechanical stress on RBCs accumulates even if the apparent cell shape recovers. The finding provides quantitative insights for the systematic comparison among various exisiting measurement methods of mechanical responses of cells.

An on-chip deformability checker is proposed to improve the velocity-deformation correlation for red blood cell (RBC) evaluation. RBC deformability has been found related to human diseases, and can be evaluated based on RBC velocity through a microfluidic constriction as in conventional approaches. The correlation between transit velocity and amount of deformation provides statistical information of RBC deformability. However, such correlations are usually only moderate, or even weak, in practical evaluations due to limited range of RBC deformation. To solve this issue, we implemented three constrictions of different width in the proposed checker, so that three different deformation regions can be applied to RBCs. By considering cell responses from the three regions as a whole, we practically extend the range of cell deformation in the evaluation, and could resolve the issue about the limited range of RBC deformation. RBCs from five volunteer subjects were tested using the proposed checker. The results show that the correlation between cell deformation and transit velocity is significantly improved by the proposed deformability checker. The absolute values of the correlation coefficients are increased from an average of 0.54 to 0.92. The effects of cell size, shape and orientation to the evaluation are discussed according to the experimental results. The proposed checker is expected to be useful for RBC evaluation in medical practices.

Biomembranes, which are mainly composed of neutral and charged lipids, exhibit a large variety of functional structures and dynamics. Here, we report a coarse-grained molecular dynamics (MD) simulation of the phase separation and morphological dynamics in charged lipid bilayer vesicles. The screened long-range electrostatic repulsion among charged head groups delays or inhibits the lateral phase separation in charged vesicles compared with neutral vesicles, suggesting the transition of the phase-separationmechanism from spinodal decomposition to nucleation or homogeneous dispersion. Moreover, the electrostatic repulsion causes morphological changes, such as pore formation, and further transformations into disk, string, and bicelle structures, which are spatiotemporally coupled to the lateral segregation of charged lipids. Based on our coarse-grained MD simulation, we propose a plausible mechanism of pore formation at the molecular level. The pore formation in a charged-lipid-rich domain is initiated by the prior disturbance of the local molecular orientation in the domain.

Regeneration of a tissue fragment of freshwater polyp Hydra is accompanied by significant morphological fluctuations, suggesting the generation of active forces. In this study, we utilized a two fingered micro-robotic hand to gain insights into the mechanics of regenerating tissues. Taking advantage of a high force sensitivity (similar to 1 nN) of our micro-hand, we non-invasively acquired the bulk elastic modulus of tissues by keeping the strain levels low (epsilon < 0.15). Moreover, by keeping the strain at a constant level, we monitored the stress relaxation of the Hydra tissue and determined both viscous modulus and elastic modulus simultaneously, following a simple Maxwell model. We further investigated the correlation between the frequency of force fluctuation and that of morphological fluctuation by monitoring one "tweezed" tissue and the other "intact" tissue at the same time. The obtained results clearly indicated that the magnitude and periodicity of the changes in force and shape are directly correlated, confirming that our two fingered micro-hand can precisely quantify the mechanics of soft, dynamic tissue during the regeneration and development in a non-invasive manner. (C) 2016 AIP Publishing LLC.

<p>細胞骨格のダイナミクスは，細胞の示す特異な粘弾性などの起源となっており，生命現象の物理学的理解の足がかりとなる興味深い特徴である．本研究では，毛細血管を模したマイクロ流体デバイス中で赤血球を高速かつ高精度に操作することにより，細胞骨格の動的組み換わり（リモデリング）が関わる変形挙動を引き出した．特に，細胞全体の長さスケールに渡るリモデリングは既知の局所的な細胞骨格ダイナミクスに比べ3桁程度遅いことがわかった．</p>

An automatic system for evaluating single cell viscoelasticity is proposed and tested in this paper. The system includes three main operations, and they are the operations of catch, load and launch. In the catch operation, the system is capable of capturing a target cell by high-speed vision and high-frequency flow control. The captured cell is pushed into a narrow constriction for the load operation. Different durations of loading time are applied to cells for evaluating cell viscoelasticity. Finally, the cell is launched out from the constriction, and the recovery response of the cell is monitored for cell characterization. Human red blood cells are experimentally tested by the proposed system. The experimental results show that the system successfully perform the evaluation, and the viscoelastic characteristics of the cells are discussed.

A high-resolution cell manipulation system is presented for investigating red blood cell deformation under longstanding load in this paper. Because the low Reynolds number in microfluidic system, cell position can be manipulated by controlling the flow in a microchannel. A high-speed vision system is embedded in the system for providing cell present position as the feedback signal for the controller while a syringe pump actuated by a piezoelectric actuator is employed for flow control in the channel. The system is utilized for applying longstanding load on human red blood cells. The longstanding load is generated by manipulating a cell into a constriction channel where the cross-sectional size is smaller than the size of the cell. The cell has to deform due to the geometrical constraints of the constriction. Both the system performance and cell response to longstanding load have been evaluated. The manipulation system successfully achieves cell positioning as accurate as 0.24 mu m while red blood cells are found always exponentially shrink with respect to time, and an average shrinkage of 1.82 mu m in 5 minutes is observed. Details of system construction and discussion on the cell response are presented.

Active force generation in living organisms, which is mainly involved in actin cytoskeleton and myosin molecular motors, plays a crucial role in various biological processes. Although the contractile properties of actomyosin have been extensively investigated, their dynamic contribution to a deformable membrane remains unclear because of the cellular complexities and the difficulties associated with in vitro reconstitution. Here, by overcoming these experimental difficulties, we demonstrate the dynamic deformation of a reconstituted lipid interface coupled with self-organized structure of contractile actomyosin. Therein, the lipid interface repeatedly oscillates without any remarkable periods. The oscillatory deformation of the interface is caused by the aster-like three-dimensional hierarchical structure of actomyosin inside the droplet, which is revealed that the oscillation occurs stochastically as a Poisson process.

Actomyosin actively generates contractile forces that provide the plasma membrane with the deformation stresses essential to carry out biological processes. Although the contractile property of purified actomyosin has been extensively studied, to understand the physical contribution of the actomyosin contractile force on a deformable membrane is still a challenging problem and of great interest in the field of biophysics. Here, we reconstitute a model system with a cell-sized deformable interface that exhibits anomalous curvature-dependent wrinkling caused by the actomyosin cortex underneath the spherical closed interface. Through a shape analysis of the wrinkling deformation, we find that the dominant contributor to the wrinkled shape changes from bending elasticity to stretching elasticity of the reconstituted cortex upon increasing the droplet curvature radius of the order of the cell size, i.e., tens of micrometers. The observed curvature dependence is explained by the theoretical description of the cortex elasticity and contractility. Our present results provide a fundamental insight into the deformation of a curved membrane induced by the actomyosin cortex.

We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids. We observed the membrane morphologies by fluorescent and confocal laser microscopy in lipid mixtures consisting of a neutral unsaturated lipid [dioleoylphosphatidylcholine (DOPC)], a neutral saturated lipid [dipalmitoylphosphatidylcholine (DPPC)], a charged unsaturated lipid [dioleoylphosphatidylglycerol (DOPG((-)))], a charged saturated lipid [dipalmitoylphosphatidylglycerol (DPPG((-)))], and cholesterol (Chol). In binary mixtures of neutral DOPC-DPPC and charged DOPC-DPPG((-)), spherical vesicles were formed. On the other hand, pore formation was often observed with GUVs consisting of DOPG((-)) and DPPC. In a DPPC-DPPG((-)-)Chol ternary mixture, pore-formed vesicles were also frequently observed. The percentage of pore-formed vesicles increased with the DPPG((-)) concentration. Moreover, when the head group charges of charged lipids were screened by the addition of salt, pore-formed vesicles were suppressed in both the binary and ternary charged lipid mixtures. We discuss the mechanisms of pore formation in charged lipid mixtures and the relationship between phase separation and the membrane morphology. Finally, we reproduce the results seen in experimental systems by using coarse-grained molecular dynamics simulations.

We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2 mL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems.

In this study, we physically modeled the influence of endotoxin-induced sepsis symptoms on human red blood cells (RBCs) by quantifying the impact of endotoxins on the cell mechanics by the analysis of Fourier-transformed mean square amplitude of shape fluctuation, called flicker spectroscopy. With the aid of a microfluidic diffusion chamber, we noninvasively determined principal mechanical parameters of human RBCs in the absence and presence of endotoxins for individual RBCs for the first time. Because of the elongation of saccharide chain length of endotoxins, we found an increase in the morphological transition from discocytes to echinocytes, and monotonic changes in the mechanical parameters. Since septic shocks often cause lethal risks of neonates, we measured the mechanical parameters of neonatal RBCs, and compared them to those of adult RBCs. The quantitative comparison reveals that neonatal RBCs are more susceptible to the effect of endotoxins than adult RBCs. Furthermore, coincubation with the antiseptic peptide P19-2.5 (Aspidasept) with endotoxin results in a slight suppression of the impact of the endotoxin. The strategy proposed in our study can potentially be applied for the quantitative diagnosis of RBCs based on mechanical readouts.

Recently, the transfer method has been shown to be useful for preparing cell-sized phospholipid bilayer vesicles, within which desired substances at desired concentrations can be encapsulated, with a desired asymmetric lipid composition. Here, we investigated the transfer process of water-in-oil (W/O) droplets coated by phospholipid monolayers across an oil/water interface by both experimental observation and theoretical modeling. Real-time experimental observation of the transfer revealed that the transfer process is characterized by three kinetic regimes: a precontact process (approaching regime), an early fast process (entering regime), and a late slow process (relaxation regime). In addition, bigger droplets require much more time to transfer than smaller droplets. We propose a theoretical model to interpret this kinetic process. Our theoretical model reproduces the essential aspects of the transfer kinetics, including its size-dependence.

The spontaneous formation of a thread-like pattern with negatively charged lipids on an oil/water interface is reported. An analysis of the time-dependent change at the interface observed by fluorescence microscopy revealed that the thread-like pattern is generated through a two-step mechanism. First, inverted lipid micelles in the bulk-oil phase gradually diffuse onto the oil/water interface. Next, the micelles are adsorbed on the interface and self-assemble to form the thread-like pattern. The essential characteristics of this pattern formation are theoretically reproduced by a simple Monte Carlo simulation that takes into account the kinetics in the coalescence of charged micelles on a 2D interface. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4722079]