Microfluidics technology is a hot topic in biochemistry because it can not only unlock new types of sensors and lab-on-a-chip devices, but also make experiments such as drug testing more efficient. Whereas most of microfluidics focuses on the steady flow of small volumes of water through channels, some scientists have found much value in carefully manipulating and mixing individual droplets.
Droplets can play the part of small reaction chambers where chemicals are mixed, and they can also be used as a medium for cell cultures. At Ritsumeikan University, Japan, Professor Satoshi Konishi and colleagues have been recently perfecting various techniques for the precise manipulation of droplets in what’s known as droplet-array sandwiching technology (DAST). Put simply, DAST consists of orderly laying out droplets in top and bottom arrays on two separate flat surfaces. These surfaces are made to face each other and then brought together close enough so that droplets make contact and mix, partially exchanging their contents.
In a past study, Professor Konishi and colleagues came up with a clever way to control the height of individual droplets in DAST through a technique called electrowetting-on-dielectric (EWOD). By applying a voltage to droplets laid over a hydrophilic–hydrophobic pattern, they could make them slightly shorter on demand. In turn, this enabled them to select which droplet pairs should make contact when the top and bottom DAST surfaces were brought close together. On top of the versatility EWOD adds to batch operations, it provides a very simple way to control the contact time between droplet pairs.
This last point proved to be instrumental to extend the benefits and potential applications of DAST even further, as Professor Konishi and two more researchers from Ritsumeikan University showed in a more recent study. It turns out that the equalization of chemical concentrations between the top and bottom droplets does not occur instantaneously, but over time. By adjusting the contact time between individual droplet pairs through EWOD, the researchers were able to decide the final concentrations of chemicals in the droplets. The team applied this idea to accurately control the chemical stimulation provided to droplet cell cultures, demonstrating the great potential DAST holds for drug screening and cell-based analysis. This paper was made available online on July 26, 2022, and it was published in Volume 370 of Sensors and Actuators B: Chemical on November 1, 2022.
First, the researchers cultured HeLa cells, a widely studied cancer cell line, on an adhesive substrate within 2 mm droplets. Then, the droplet’s medium was changed to one containing Fluo-3 AM. This staining chemical indicates the presence of calcium ions via fluorescence. Using DAST, the researchers finally mixed the cell culture droplets with histamine-containing droplets, carefully controlling the contact time between them to obtain the desired histamine concentration in the cell cultures.
To demonstrate that the correct amount of histamine was supplied to each culture, the researchers visualized the calcium oscillations in the cells using a fluorescence microscope, taking advantage of the Fluo-3 AM probes. The idea is that histamine acts as a stimulant to HeLa cells, increasing the amplitude of their calcium oscillations. “We chose to study cellular calcium oscillations because they are a well-known research subject related to several vital cellular processes in neurons, bones, skeletal muscles, the liver, and more,” explains Professor Konishi, “Thus, evaluating calcium signaling is an important subject for biological research.”
As expected, the contact time between droplets was directly related to the amplitude of the observed calcium oscillations, implying that longer contact times led to more histamine making its way from the top droplet to the bottom one (which contained the cell culture). In turn, this means that DAST, in combination with EWOD, has the potential to precisely control the concentration of substances in droplets.
The results of this study could have tremendous implications for biochemical science and applications, since DAST can fundamentally change the way experiments in liquids are conducted. “Droplets in DAST can not only act as reaction chambers or cell cultures, but also fulfill the roles of liquid-handling tools such as pipettes on a much smaller scale,” remarks Professor Konishi. “We anticipate that our technology using individually controlled droplet arrays will become a useful and efficient tool for cell-based screening owing to its high-throughput performance.”
Watch out for when these tiny tools finally make their way to biochemistry laboratories worldwide!
Authors: Satoshi Konishi1,3,4, Yuriko Higuchi2, Asuka Tamayori3
1Department of Mechanical Engineering, College of Science and Engineering, Ritsumeikan University
2Graduate School of Pharmaceutical Sciences, Kyoto University
3Graduate Course of Science and Engineering, Ritsumeikan University
4Ritsumeikan Global Innovation Research Organization, Ritsumeikan University
About Ritsumeikan University, Japan
Ritsumeikan University is one of the most prestigious private universities in Japan. Its main campus is in Kyoto, where inspiring settings await researchers. With an unwavering objective to generate social symbiotic values and emergent talents, it aims to emerge as a next-generation research university. It will enhance researcher potential by providing support best suited to the needs of young and leading researchers, according to their career stage. Ritsumeikan University also endeavors to build a global research network as a “knowledge node” and disseminate achievements internationally, thereby contributing to the resolution of social/humanistic issues through interdisciplinary research and social implementation.
About Professor Satoshi Konishi from Ritsumeikan University, Japan
Satoshi Konishi received BS, MS, and PhD degrees in Electronics and Electrical Engineering from the University of Tokyo, Japan, in 1991, 1993 and 1996, respectively. He joined Ritsumeikan University in 1996, where he currently serves as Professor and director of the Micro/Nano Mechatronics Laboratory. His study is devoted to microelectromechanical systems (MEMS), covering a broad range of topics from fundamental to applied fields. His current research focuses on biomedical MEMS, especially multiscale interfaces in biomedical engineering.
This study was partially supported by the Ritsumeikan Global Innovation Research Organization.
Sensors and Actuators B Chemical
Method of Research
Subject of Research
Title of original paper: Cellular calcium oscillations in droplets with different chemical concentrations supplied by droplet-array sandwiching technology
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.