Single-Cell Millifluidics: a yet overlooked potential for single-cell research

Single-Cell Millifluidics: a yet overlooked potential for single-cell research

Microfluidic technologies, using chips and cartridges with channels < 100 µm in width, have been all the craze in recent years and imposed themselves as a method of reference for single-cell analysis. However, narrow channel sizes and tiny reaction volumes are not always optimal conditions to work with, depending on the goal of your studies. The automation of millifluidic devices, this time using channels 1 mm wide, presents an alternative with strong advantages and the technology has moved forward with the recent development of devices such as the Millidrop Analyzer. We discussed all things millifluidics with Dr. Arthur Goldstein, who recently graduated from the ESPCI in Paris with a Ph.D. that included the study of the dissolution of phosphate particles by natural soil samples.


The main advantage of single-cell millifluidics lies in its easier droplet handling

With the rise of microfluidics, turning back to millifluidics could initially sound counter-intuitive, as larger droplets mean using fewer samples and lower screening efficiency.

“What you lose in screening power, you gain in usability”, explains Goldstein. “The larger the drop, the easier it is to identify. In millifluidics, each drop is identified with a number, and you can consistently monitor multiple parameters, whether they are kinetic, enzymatic, or growth curves for each droplet over time.”

“Millifluidics is the great compromise between the number of droplets, their identification, and the easy handling of those droplets.“

Using 96-well or 384-well plates, millifluidic platforms can handle hundreds to thousands of samples in parallel, but each drop can be handled individually, for example in the initial composition of each droplet or the addition of specific reagents at a certain time.

A strong advantage of the millifluidic scale is the ability to recover droplets of interest. If a droplet developed a microorganism, a strain, or a cell type with a particular phenotype or triggering a phenomenon of interest, you can easily retrieve the specific droplet for further sequencing, culture, or any downstream analysis. Droplets of interest can be dispensed in a 96-well plate and directly retrieved by hand from each well.

As a bonus and in contrast to microfluidics, millifluidic devices can be used by a technician with no special training, with the newest systems qualified as “plug and play”.

Investigating a simpler way to select a few unique cells to analyze from a large sample? Have a look at this elegant solution researchers at the university of technology of Sydney have developed using etched hydrophobic chips to selectively perform scRNA-seq on fetal cells.


The millifluidic advantages of larger channel and droplet sizes

Thanks to the larger channel size, millifluidic devices usually do not require surfactants, meaning it reduces the risk of micellar transfers between droplets. For example, some fluorescent probes would be less likely to diffuse in the oil phase and/or disseminate into other drops, contaminating samples.

Millifluidic machines can also easily insert air bubbles in-between sample droplets, providing essential resources such as oxygen for the metabolism of micro-organisms or cell sub-types such as organoids, which is trickier on microfluidic chips.

Millifluidics droplets are regularly mixed, usually through friction against the channel, meaning their content is more homogeneous droplet-wide compared to microfluidic flow cells.

In terms of read-outs of activity, some fluorescent probes might be hard to detect at the microfluidic scale under the microscope, while the increased signal at the millifluidic scale makes this much easier.

“I was looking at the dissolution of insoluble phosphate particles from the secretion of soil microorganisms”, explains Goldstein. “I measured turbidity and it would not work at the microfluidic scale, as the properties of light would not apply on such a thin chip. In contrast, the MilliDrop machine for example can read absorbance for a large set of fluorescent probes. We were able to generate 600 drops from 60 wells, with 10 drops per well, and thousands of curves per experiment.”


Millifluidics enables a large array of options for individual cell cultures and inter-species relationships

Sometimes in single-cell studies, it might be very advantageous to set up cultures starting from isolated cells to increase the amount of material available for single-cell analysis. Millifluidics technology is particularly suited for this purpose, as hundreds of cultivation conditions can be set up in parallel and regularly fed nutrients for continuous growth.

For individual cultures, a larger droplet volume also means a longer timeframe and a higher number of divisions before reaching saturation, letting enough time for a phenotype to develop in a population.

“Starting from a single cell, I would say you have 6 to 7 divisions in a microfluidic droplet, compared to up to 15 in millifluidics”, comments Goldstein. “You can also encapsulate up to 10 different species, create communities, and observe their cohabitation. For example, we know the hydrolysis of polymers like cellulose or plastic is the combined work of multiple microorganisms, and that they would not be able to achieve the same outcome separately. Studying such cohabitations can reveal a lot about the mechanisms of polymer degradation.”

“Another example is the degradation of atrazine, a pesticide that was formerly used before potentially hazardous properties were discovered,” continues Goldstein. “After screening, a research team discovered that only a specific combination of four different microorganisms was able to degrade this compound, and this is the kind of experiments millifluidics is perfectly suited for.”


Millifluidics can also detect the competitive behavior of microorganisms

The combination of different selected cells in a droplet could also be applied in an antagonistic manner, pitting organisms against each other. It could be applied as a low-throughput screening for the characterization of “novel” antibiotics, screening thousands of many bacterial combinations, monitor the culture growths, and observe the outcome. Droplets of interest could then be directly retrieved to run a mass spectrometry analysis and identify any novel compound.

Goldstein has tested such an application: “We wanted to evaluate the ability of a soil sample to prevent the growth of pathogens. So we added a few cells of a known phytopathogen to droplets, separately added our soil sample suspensions to those droplets, and after a while checked which sample prevented pathogen growth.”

The easy manipulation of droplets in millifluidics also enables an automatic setup for individual cultures. For example, droplets for cultivating cells can be halved at regular intervals, with one half saved for analysis and the other half mixed with fresh medium and left to grow longer. Such a setting would be ideal to measure the timeline for a microbe to develop a resistance against a particular antibiotic for example.

The future of millifluidics is bright and could nicely fit the goals of some single-cell studies. With the ongoing progress of “organ-on-a-chip” devices and 3D cell cultures, the technology is still in constant progress with new opening possibilities, as Goldstein concludes.

“Researchers have recently shown that you can monitor the electromagnetic properties of a droplet in millifluidics, which depends on the concentration of the solute and can be a very interesting parameter to complement optical measurement. As far as I know, this is something that is not possible to do at the microfluidic scale.”


Dr. Arthur Goldstein is now a postdoc at the ESPCI and you can find his Ph.D. thesis here (in French).

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