Droplet Toolbox Opens New Possibilities for Genetic and Drug Screening of Small Animals

Genetic screens and drug screens play an essential role in the understanding of gene functions and the development of therapeutics. Genetic screens can identify genes contributing to a defect or disease state, while drug screens search for treatments that can restore normal function.

Traditionally, large-scale screens have been performed on single cells because of their small size and cost-efficiency, but with the limitation of missing the complexity of whole animals.

Screening animals allows for tackling phenotypes more relevant to human diseases, in particular dynamic phenotypes such as brain activity, muscle activity, or behavior. Such screens present huge benefits for the study of neuropsychological disorders and behavioral disorders. However, the challenge is that handling animals is far more challenging than handling single cells  – especially when the need is to scale up to the thousands.

A team of researchers led by Hang Lu, a professor in Georgia Tech’s School of Chemical and Biomolecular Engineering, published a paper in the journal Small on a technological platform that would solve this bottleneck using droplet microfluidics.

Encapsulating microscopic animals in tiny droplets transposes the problem into a different perspective. Instead of manipulating animals directly, one now aims at handling a “passive” object containing an animal, Lu said. Working with droplets also has several advantages: no cross contamination, small volumes, and a gentle way of trapping and transporting the animal.

“Using droplet microfluidics allows for processing animals faster, user bias-free, and with nanoliter amounts of reagents. This platform is ideal to study the animal reactions to dynamic alterations of its chemical environment,” Lu said.

Studying C. elegans

As a proof of application, the team worked with C. elegans, a freely living nematode that varies in size from a couple hundred microns at the larval stage to one millimeter once adult. C. elegans is a widely used model organism in genetics that was first introduced in the 1960s. Several Nobel Prizes have been rewarded to researchers working on this model.

Besides having its genome fully sequenced and connectome fully determined, this mainly-hermaphroditic nematode has a large progeny, short reproduction cycle, and is inexpensive to maintain. Altogether, these attributes have established C. elegans as a key organism for large-scale screens. Traditionally, the screens have been performed manually and required months, if not years, of graduate student time and been often limited to somewhat simple animal responses.

The droplet platform may speed up that process as well as broaden the scope of assays performed on the animals. “We have developed a droplet toolbox that allows us to manipulate the animals and their environment and perform elaborate automated protocols” explained Guillaume Aubry, a research scientist in the Lu lab.

The team members first created an animal encapsulation system where each animal is packaged in an aqueous droplet. Then they created various droplet manipulators to alter the animal’s environment. In particular, a liquid exchanger allows researchers to swap the animal from one droplet to another. Finally, they established a method for programming protocols and performing tasks automatically.

Increased Throughput, Applications

The researchers demonstrated a throughput of more than a hundred animals per hour for cases of observing the animal behavior in response to a chemical stimulant. They also showed that this platform can be used to monitor brain activity simultaneously, which opens countless applications in neuroscience to decipher neuronal circuitry, dynamics, and correlate brain activity with behavior.

The team explained that the platform is adaptable to other small animals in the micro- to millimetric range. Because the design principle relies on droplets, the system is scalable to accommodate the animal size. As an example, the team scaled down the system to work with the first larval stage of C. elegans, which is considerably smaller, and demonstrated monitoring the larvae’s neuronal activity to a sudden change of chemical environment.

“Ultimately this platform will help to bridge the gap between environment, genotype, and phenotype,” Aubry said. “The platform opens up exciting possibilities in particular for the study of neurodevelopmental disorders. With this scalable tool, one can envision monitoring the development of single individuals and assessing their behavioral and neuronal activities over time. Another exciting avenue is to take further advantage of the nanoliter volumes to pursue drug screens, for example on the effect of neuronal activity.”

Funding

The authors acknowledge National Institute of Health (NIH R21NS117066, R01NS096581, and R01AG056436) for funding. This work was performed in part at the Georgia Tech Institute for Electronics and Nanotechnology, a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant ECCS-1542174).

Citation

G. Aubry, M. Milisavljevic, and Hang Lu “Automated and Dynamic Control of Chemical Content in Droplets for Scalable Screens of Small Animals” Small 18, 2200319 (2022) DOI: 10.1002/smll.202200319 https://onlinelibrary.wiley.com/doi/full/10.1002/smll.202200319 (Supplemental Movie 5 and Supplemental Movie 7 of the Supporting Information of the paper)