Scientists showcase how the concentration of chemicals in droplets can be efficiently controlled by running experiments in the tiniest of cell cultures.

Droplet-array sandwiching technology (DAST) lets one use droplets as tiny reaction chambers or cell culture media. In a recent study, scientists demonstrated how novel techniques to control the mixing of these droplets could be used to finely adjust the concentration of chemicals in them. Through representative experiments on cellular stimulation, the researchers showcased how DAST could become an essential high-throughput tool for biochemistry, paving the way to more effective drug screening and cell-based analysis.

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: Chemicalon 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!

Title of original paper: Cellular calcium oscillations in droplets with different chemical concentrations supplied by droplet-array sandwiching technology
Journal: Sensors and Actuators B: Chemical
DOI: 10.1016/j.snb.2022.132435
Authors: Satoshi Konishi^1,3,4,*, Yuriko Higuchi², Asuka Tamayori^3
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 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.

Funding information

This study was partially supported by the Ritsumeikan Global Innovation Research Organization.