Shuichi Yamaguchi

Cell patterning through inkjet printing of one cell per droplet

The inkjet ejection technology used in printers has been adopted and research has been conducted on manufacturing artificial tissue by patterning cells through micronozzle ejection of small droplets containing multiple cells. However, stable injection of cells has proven difficult, owing to the frequent occurrence of nozzle clogging. In this paper, a piezoelectric inkjet head constructed with a glass capillary that enabled viewing of the nozzle section was developed, the movement of cells ejected from the nozzle tip was analyzed, and a method for stably ejecting cells was verified. A pull-push ejection method was compared with a push-pull ejection method regarding the voltage waveform applied to the piezoelectric element of the head. The push-pull method was found to be more suitable for stable ejection. Further, ejection of one cell per droplet was realized by detecting the position of the cell in the nozzle section and utilizing these position data. Thus, a method for more precise patterning of viable cells at desired position and number was established. This method is very useful and promising not only for biofabrication, 3D tissue construction, cell printing, but also for a number of biomedical application, such as bioMEMS, lab on a chip research field.

Piezoelectric inkjet-based one cell per one droplet automatic printing by image processing

Piezoelectric inkjet printer technology recently has gained attention and was utilized eject particles and cells of several tens of μm scale, which is large compared to colloid used in printer ink. However, the inkjet head was originally design for color ink. Therefore, problems, such as, head clogging and trajectory error occurred. In this study, those problems were addressed, and optimal condition for stable cells ejection was verified. Furthermore, one cell per one droplet printing method was established by observing cells position on the inside of inkjet head. Finally, automatic one cell per one droplet printing system was successfully developed by processing image of the inkjet head to automatically detect cells position. The automatic one cell per one droplet printing system was successfully developed with 98% successful ratio.

Influence of droplet size to cell viability in flash freezing method

We examine cell cryopreservation method without cryoprotectant agent (CPA). In general, the CPA is supplemented to the freezing medium to inhibit the generation and growth of ice crystals inside and outside the cell because cell structures are destroyed by the ice crystals. On the other hand, we need to think about the cytotoxicity of CPA in the cell cryopreservation. The faster the cooling rate become, the more the generation and growth of ice crystals are inhibited inside and outside the cell. To freeze picolitter droplets at high cooling rate, we used inkjet technique and liquid nitrogen. Thereby, the damage to the cells was inhibited. The viability of the flash frozen cells at 20 pL improved about 60 times higher than the viability of frozen cells by slow-rate freezing without CPA. We confirmed that the cell viability of cryopreservation method without CPA increased in flash freezing. Moreover, the viability of the flashly frozen cells at 20 pL increased to more than twice the viability of the flash frozen cells at 200 pL. As the droplet size became small, the cell viability increased in the flash freezing method. We were able to find the possibility of the inkjet-based flash cell freezing toward cryopreservation without CPA.

Rapid Single Cell Printing by Piezoelectric Inkjet Printer

Tissue engineering is rapidly developing to assist in the treatment of organ loss or tissue damage. On the other hand, the relatively long process of drug development may also need in vitro fabricated organ for drug testing, which opens up faster door to development process. Several methods and technologies have been introduced as valuable tools for tissue engineering, and inkjet printing stands out as the “scaffold-less” tissue engineering tools starting from the early 2000s, although the scaffold-less feature is still debatable. Furthermore, this technology was also used to encapsulate single cells in the fluid droplet, thus opening up the possibility for isolation of single cells in the open space and better spatiotemporal control of cell printing at individual level. Combined with the high-frequency droplet ejection capability of inkjet printer, high-throughput single cells array and high-speed fabrication of finer detail tissue fabrication may also be possible in the future. This chapter introduced several methods used to deposit cells on arbitrary pattern and explored on the challenge in the implementation of inkjet printing technology to deposit cells and single cells on arbitrary pattern. This chapter also argued several adjustments and limitations for relatively large particles and cell printing, which are in a different size scale compared to ink particles in the commercial inkjet printer.

Ejection of a single cell in a single droplet using piezoelectric inkjet head

The inkjet ejection technology used in printers has been adopted and research has been conducted on manufacturing artificial tissue through micronozzle ejection of small droplets containing multiple cells by patterning them. However, stable injection of cells has proven difficult, owing to the frequent occurrence of nozzle clogging. Almost no research has been conducted on a method for driving inkjet heads for the purpose of stably ejecting cells. In the present research, a glass capillary that enabled viewing of the nozzle section was used, and a piezoelectric inkjet head was developed, the movement of cells ejected from the nozzle tip was analyzed, and a method for stably ejecting cells was verified. In addition, the pull-push method was compared with the push-pull method for the voltage waveform applied to the piezoelectric element of the head when the cells were ejected. The push-pull method was found to be more suitable for their stable ejection. Further, a state where one droplet of ejected fluid contained only one cell was realized by detecting the position of the cell in the nozzle section and utilizing these position data. Thus, a method for more precise patterning at targeted positions was established.