Akiyuki Hasegawa

Vision-Based Automated Single-Cell Loading and Supply System

Automated continuous individual cell transfer is a critical step in single-cell applications using microfluidic devices. Cells must be aspirated gently from a buffer before transferring to operation zone so as not to artificially perturb their biostructures. Vision-based manipulation is a key technique for allowing nondestructive cell transportation. In this paper, we presented a design for an automated single-cell loading and supply system that can be integrated with complex microfluidic applications for examining or processing one cell at a time such as the current nuclear transplantation method. The aim of the system is to automatically transfer mammalian donor (~ 15 ¿m) or oocyte (~ 100 ¿ m) cells one by one from a container to a polydimethylsiloxane (PDMS) microchannel and then transport them to other modules. The system consists of two main parts: a single-cell suction module, and a PDMS-based microfluidic chip controlled by an external pump. The desired number of vacuumed cells can be directed into the microfluidic chip and stored in a docking area. From the batch, they can be moved to next module by activating pneumatic pressure valves located on two sides of the chip. The entire mechanism is combined with monitoring systems that perform detection/tracking and control.

Rapid fabrication system for three-dimensional tissues using cell sheet engineering and centrifugation.

Three-dimensional (3D) tissues can be reconstructed by cell sheet technology, and various clinical researches using these constructed tissues have already been initiated to regenerate damaged tissues. While 3D tissues can be easily fabricated by layering cell sheets, the attachment period for cell adhesion between a cell sheet and a culture dish, or double-layered cell sheets normally takes 20-30 min. This study proposed a more rapid fabrication system for bioengineered tissue using cell sheet technology and centrifugation. A C2C12 mouse myoblast sheet harvested from a temperature-responsive culture dish will attach tightly to a culture dish or another cell sheet at 37°C after a 20 min-incubation. However, the same cell sheet centrifuged (12-34 × g) for 3 min also attached tightly to a dish or another cell sheet at 37°C after only a 3 min-incubation. The manipulation time was reduced by approximately two-thirds by centrifugation. The rapid attachments were also cross-sectionally confirmed by optical coherence tomography. These rapidly constructed cell sheet-tissues using centrifugation showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor, like those prepared by the conventional method; indicating complete cell sheet-attachment without any cell damage. This new system will be a powerful tool in the fields of cell sheet-based tissue engineering and regenerative medicine, and accelerate the use of cell sheets in clinical applications.

Multiple cell suction and supply system for automated cell manipulation on microfluidic channel

Advances in biology have clarified the details of many phenomena and their results have been applied to many fields. However, in these fields, applications require dexterous and skillful manipulation. Hence, realizing high productivity is still difficult. To solve this problem, assisted cell manipulation and automated systems have been researched and microfluidic channel technology has been used in these fields. Most research focuses on only one part of the cell manipulation process, such as the cutting or sorting of cells. In the meantime, less attention has been paid to methods of supplying the target cells to a microfluidic channel. We have developed a multiple cell supply system for the microfluidic channel to realize automated cell cloning. This system sucks bovine egg cells with a size of 100[µm] and sends them to the microfluidic channel, then finally sends them to the cell manipulation part. It consists of a microfluidic channel, a pump, solenoid valves, camera systems, and an automatic stage. The cell supply action can be realized by controlling these parts through a computer program.

Detection sensor for flowing particles in micro channel

In this paper, we propose a sensor for detecting particles flowing in a micro channel fabricated with polydimethylsiloxane (PDMS) chip. A light source placed at one side of a micro channel irradiates the micro channel through plastic optical fibers (POF), and a detector placed at the opposite side of the micro channel receives light transmitted through the POF. Since this sensor simultaneously detects plural particles with a diameter of around 100 µm flowing in a micro channel, it can be implemented in automatic measurement systems of cells in desktop bio plant, on which automated embryonic cell manipulation is achieved using micro robotics technology.

Distribution and Translocation of Photoreceptor Gβγ-phosducin System in Medaka Retina‡

Unlike other vertebrates, teleosts have rod‐ and cone‐specific phosducins (PD‐R and PD‐C) in the retina. To evaluate the teleost Gβγ‐PD systems, we isolated cDNAs encoding medaka Gβ1 and GβC, which selectively expressed rods and cones. Immunohistochemical studies showed that the strong reactivity of GβC but not PD‐C was detected in cone outer segments. In rod outer segments (ROS), PD‐R reactivity was stronger in light‐adapted retina than in dark‐adapted retina. Western blot analyses of fractions torn from the cryosections showed that the PD‐R concentration was low in dark‐adapted ROS. It is suggested that PD‐R is translocated to ROS and effectively downregulates the phototransduction cascade in light‐adapted rods.

Optical coherence microscopy of living cells and bioengineered tissue dynamics in high-resolution cross-section

Optical coherence tomography (OCT) is a valuable tool in the cross-sectional observation/analysis of three-dimensional (3-D) biological tissues, and that histological observation is important clinically. However, the resolution of the technology is approximately 10-20 μm. In this study, optical coherence microscopy (OCM), a tomographic system combining OCT technology with a microscopic technique, was constructed for observing cells individually with a resolution at the submicrometer level. Cells and 3-D tissues fabricated by cell sheet technology were observed by OCM. Importantly, the cell nuclei and cytoplasm could be clearly distinguished, and the time-dependent dynamics of cell-sheet tissues could be observed in detail. Additionally, the 3-D migration of cells in the bioengineered tissue was also detected using OCM and metal-labeled cells. Bovine aortic endothelial cells, but not NIH3T3 murine embryonic skin fibroblasts, actively migrated within the 3-D tissues. This study showed that the OCM system would be a valuable tool in the fields of cell biology, tissue engineering, and regenerative medicine.

Three-Dimensional Human Cardiac Tissue Engineered by Centrifugation of Stacked Cell Sheets and Cross-Sectional Observation of Its Synchronous Beatings by Optical Coherence Tomography

Three-dimensional (3D) tissues are engineered by stacking cell sheets, and these tissues have been applied in clinical regenerative therapies. The optimal fabrication technique of 3D human tissues and the real-time observation system for these tissues are important in tissue engineering, regenerative medicine, cardiac physiology, and the safety testing of candidate chemicals. In this study, for aiming the clinical application, 3D human cardiac tissues were rapidly fabricated by human induced pluripotent stem (iPS) cell-derived cardiac cell sheets with centrifugation, and the structures and beatings in the cardiac tissues were observed cross-sectionally and noninvasively by two optical coherence tomography (OCT) systems. The fabrication time was reduced to approximately one-quarter by centrifugation. The cross-sectional observation showed that multilayered cardiac cell sheets adhered tightly just after centrifugation. Additionally, the cross-sectional transmissions of beatings within multilayered human cardiac tissues were clearly detected by OCT. The observation showed the synchronous beatings of the thicker 3D human cardiac tissues, which were fabricated rapidly by cell sheet technology and centrifugation. The rapid tissue-fabrication technique and OCT technology will show a powerful potential in cardiac tissue engineering, regenerative medicine, and drug discovery research.

Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge

Confluent cultured cells on a temperature‐responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three‐dimensional (3‐D) tissue can be fabricated by the layering of cell sheets. A resulting 3‐D multilayered cell sheet‐tissue on a temperature‐responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3‐D multilayered constructs, we attempted to layer cell sheets on a temperature‐responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22‐23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36‐37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet‐layering process. The heating centrifugation shortened the fabrication time by one‐fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue‐fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy.

Micro valve system for individual cell transportation in microfluidic chip

Recently, micro valves to control some kinds of fluids in channels in a microchip have been developed. Now we pay attention to in-chip somatic cell cloning with microfluidic technology. There are some steps, cell supply, cutting, sorting, coupling, fusion and so on, for this work. And we focused on a cell supply system spending cells one by one in microfluidic chip, and attempt to transport an individual cell to objective course by the control of pressure valves. Here, we made three kinds valves, a) open channel 2-layer valve, b) open channel 3-layer valve, and c) close channel 2-layer valve. We compare these valves about interception condition of micro channel flow and time to take for open/close. And then we evaluated them with suitable for the smooth cell transportation by these comparisons.

Rapid creation system of morphologically and functionally communicative three-dimensional cell-dense tissue by centrifugation: Rapid creation of three-dimensional cell-dense tissue by centrifugation

This study reports a rapid fabrication system of a morphologically and functionally communicative three‐dimensional (3D) cell‐dense tissue without scaffolds by centrifugation. The tight adhesion between C2C12 myoblasts and culture surface was accelerated without significant cell damage by centrifugation (80 x g, 37 °C, 30 min). A thicker tissue created on a temperature‐responsive culture surface was harvested by decreasing temperature. The 3D myoblast tissues having approximately 200 μm‐thickness were created at 1.5 h [centrifugation (80 x g, 37 °C) for 30 min and tissue harvest for 1 h]. However, in the case of without centrifugation, the myoblast tissues had fragile parts even at 7.5 h after the incubation. Additionally, electrically/functionally communicative and thicker human induced pluripotent stem (iPS) cell‐derived cardiac tissues were created rapidly by the centrifugation and cultivation at 37 °C. We report a centrifugation system that significantly shortens the creation time of 3D tissues. We envision that this procedure will contribute to the field of tissue engineering and regenerative medicine.