Shinya Sakuma

A New Dimensionless Index for Evaluating Cell Stiffness-Based Deformability in Microchannel

This paper proposes a new index for evaluating the stiffness-based deformability of a cell using a microchannel. In conventional approaches, the transit time of a cell through a microchannel is often utilized for the evaluation of cell deformability. However, such time includes both the information of cell stiffness and viscosity. In this paper, we eliminate the effect from cell viscosity, and focus on the cell stiffness only. We find that the velocity of a cell varies when it enters a channel, and eventually reaches to equilibrium where the velocity becomes constant. The constant velocity is defined as the equilibrium velocity of the cell, and it is utilized to define the observability of stiffness-based deformability. The necessary and sufficient numbers of sensing points for evaluating stiffness-based deformability are discussed. Through the dimensional analysis on the microchannel system, three dimensionless parameters determining stiffness-based deformability are derived, and a new index is introduced based on these parameters. The experimental study is conducted on the red blood cells from a healthy subject and a diabetes patient. With the proposed index, we showed that the experimental data can be nicely arranged.

Fabrication and Application of 3-D Magnetically Driven Microtools

In this paper, we describe a novel method of fabricating polymeric 3-D magnetically driven microtools (MMTs) for performing nonintrusive and contamination-free experiments on chips. In order to obtain precise and complicated 3-D patterns from magnetically driven 3-D microtools, a grayscale photolithography technique was applied by making good use of a thick negative photoresist as a sacrifice mold. By controlling the amount of ultraviolet light with a gradation of gray-tone mask, we fabricated a smoothly curved (100-¿ m gap) object without steps, which tend to appear in the case of conventional layer-by-layer photolithography techniques. A wide range of on-chip applications of microactuators can be realized by using the softness of the polymer-based 3-D MMT. For example, a microfilter and a microloader were successfully operated by a combination of magnetic and fluidic forces. The finite element method analysis of flow showed that a rotation of the 3-D MMT produces a relatively strong downward axial flow, which prevents particles from stagnating on the surface of the MMT. The produced 3-D MMT can be applied to complex on-chip manipulations of sensitive materials such as cells.

Magnetically Modified Soft Micro Actuators for Oocyte Manipulation

We have developed novel magnetically driven polymeric microtool for non-intrusive and no contamination experiments on a chip. The composite is formed by suspending magnetite particles in polydimethylsiloxane. In order to obtain precise and complicated pattern of magnetic microtools, a photolithography techniques has been applied by making good use of thick KMPR-1050 photoresist as sacrifice-mold. The novelties of these tools are 1. fabrication of any 2D shape, 2.softness, 3. no contact actuation, 4. mass production with low cost. These versatile magnetic mirotools can be applied to various functions such as stirrer, valve, loader and sorter and so on. The potential impact of this technology includes sample selection and separation, particle loading and immobilization, genetic operation, tracking, mixing and reaction techniques into portable microfluidic labs-on-a-chip, culture systems.

On-Chip Method to Measure Mechanical Characteristics of a Single Cell by Using Moiré Fringe

We propose a method to characterize the mechanical properties of cells using a robot-integrated microfluidic chip (robochip) and microscopy. The microfluidic chip is designed to apply the specified deformations to a single detached cell using an on-chip actuator probe. The reaction force is simultaneously measured using an on-chip force sensor composed of a hollow folded beam and probe structure. In order to measure the cellular characteristics in further detail, a sub-pixel level of resolution of probe position is required. Therefore, we utilize the phase detection of moire fringe. Using this method, the experimental resolution of the probe position reaches 42 nm. This is approximately ten times smaller than the optical wavelength, which is the limit of sharp imaging with a microscope. Calibration of the force sensor is also important in accurately measuring cellular reaction forces. We calibrated the spring constant from the frequency response, by the proposed sensing method of the probe position. As a representative of mechanical characteristics, we measured the elastic modulus of Madin-Darby Cannie Kidney (MDCK) cells. In spite of the rigid spring constant, the resolution and sensitivity were twice that achieved in our previous study. Unique cellular characteristics can be elucidated by the improvements in sensing resolution and accuracy.

A Single Cell Extraction Chip Using Vibration-Induced Whirling Flow and a Thermo-Responsive Gel Pattern

We propose a single cell extraction chip with an open structure, which utilizes vibration-induced whirling flow and a single cell catcher. By applying a circular vibration to a micropillar array spiral pattern, a whirling flow is induced around the micropillars, and target cells are transported towards the single cell catcher placed at the center of the spiral. The single cell catcher is composed of a single-cell-sized hole pattern of thermo-responsive gel. The gel swells at low temperatures (≲32 ◦C) and shrinks at high temperatures (≳32 ◦C), therefore, its volume expansion can be controlled by an integrated microheater. When the microheater is turned on, a single cell is trapped by the hole pattern of the single cell catcher. Then, when the microheater is turned off, the single cell catcher is cooled by the ambient temperature. The gel swells at this temperature, and the hole closes to catch the single cell. The caught cell can then be released into culture wells on a microtiter plate by heating the gel again. We conducted single cell extraction with the proposed chip and achieved a 60% success rate, of which 61% cells yielded live cells.

On-chip actuation transmitter for enhancing the dynamic response of cell manipulation using a macro-scale pump

An on-chip actuation transmitter for achieving fast and accurate cell manipulation is proposed. Instead of manipulating cell position by a directly connected macro-scale pump, polydimethylsiloxane deformation is used as a medium to transmit the actuation generated from the pump to control the cell position. This actuation transmitter has three main advantages. First, the dynamic response of cell manipulation is faster than the conventional method with direct flow control based on both the theoretical modeling and experimental results. The cell can be manipulated in a simple harmonic motion up to 130 Hz by the proposed actuation transmitter as opposed to 90 Hz by direct flow control. Second, there is no need to fill the syringe pump with the sample solution because the actuation transmitter physically separates the fluids between the pump and the cell flow, and consequently, only a very small quantity of the sample is required (<1 μl). In addition, such fluid separation makes it easy to keep the experiment platform sterilized because there is no direct fluid exchange between the sample and fluid inside the pump. Third, the fabrication process is simple because of the single-layer design, making it convenient to implement the actuation transmitter in different microfluidic applications. The proposed actuation transmitter is implemented in a lab-on-a-chip system for red blood cell (RBC) evaluation, where the extensibility of red blood cells is evaluated by manipulating the cells through a constriction channel at a constant velocity. The application shows a successful example of implementing the proposed transmitter.

Arterial graft with elastic layer structure grown from cells

Shortage of autologous blood vessel sources and disadvantages of synthetic grafts have increased interest in the development of tissue-engineered vascular grafts. However, tunica media, which comprises layered elastic laminae, largely determines arterial elasticity, and is difficult to synthesize. Here, we describe a method for fabrication of arterial grafts with elastic layer structure from cultured human vascular SMCs by periodic exposure to extremely high hydrostatic pressure (HP) during repeated cell seeding. Repeated slow cycles (0.002 Hz) between 110 and 180 kPa increased stress-fiber polymerization and fibronectin fibrillogenesis on SMCs, which is required for elastic fiber formation. To fabricate arterial grafts, seeding of rat vascular SMCs and exposure to the periodic HP were repeated alternatively ten times. The obtained medial grafts were highly elastic and tensile rupture strength was 1451 ± 159 mmHg, in which elastic fibers were abundantly formed. The patch medial grafts were sutured at the rat aorta and found to be completely patent and endothelialized after 2.5 months, although tubular medial constructs implanted in rats as interpositional aortic grafts withstood arterial blood pressure only in early acute phase. This novel organized self-assembly method would enable mass production of scaffold-free arterial grafts in vitro and have potential therapeutic applications for cardiovascular diseases.

High Resolution Cell Positioning Based on a Flow Reduction Mechanism for Enhancing Deformability Mapping

The dispersion of cell deformability mapping is affected not only by the resolution of the sensing system, but also by cell deformability itself. In order to extract the pure deformability characteristics of cells, it is necessary to improve the resolution of cell actuation in the sensing system, particularly in the case of active sensing, where an actuator is essential. This paper proposes a novel concept, a “flow reduction mechanism”, where a flow is generated by a macroactuator placed outside of a microfluidic chip. The flow can be drastically reduced at the cell manipulation point in a microchannel due to the elasticity embedded into the fluid circuit of the microfluidic system. The great advantage of this approach is that we can easily construct a high resolution cell manipulation system by combining a macro-scale actuator and a macro-scale position sensor, even though the resolution of the actuator is larger than the desired resolution for cell manipulation. Focusing on this characteristic, we successfully achieved the cell positioning based on a visual feedback control with a resolution of 240 nm, corresponding to one pixel of the vision system. We show that the utilization of this positioning system contributes to reducing the dispersion coming from the positioning resolution in the cell deformability mapping.

On-chip enucleation of an oocyte by untethered microrobots

We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

Phase decomposition of a cell passing through a μ-channel: A method for improving the evaluation of cell stiffness

Two-phase motion of a cell inside a μ-channel is observed by the high-speed vision system. Two phases are defined as the phase of deformation and the phase of constant shape according to their characteristics. Red blood cells were used for experimental validation, and a mechanical model consisting of a spring and a damper in parallel is utilized for interpreting the behavior of the RBCs. An analysis method for acquiring the transition point between two phases is proposed, and is utilized in the analysis of the experimental results presented in this paper. Two different initial velocities of red blood cells approaching the μ-channel were used in the experiment. The experimental results show that the red blood cells with the higher initial velocity require longer distance to reach the steady state comparing to the ones with the lower initial velocity.