Automated and Dynamic Control of Chemical Content in Droplets for Scalable Screens of Small Animals

Abstract

the limited ability to change the environment and 3D animal motion make it difficult to assess dynamic phenotypes other than rough motion analysis. In contrast, microfluidics, with relevant length scales down to the animal's size, offers improved means to handle small animals and control their environment. [10][11][12][13][14] Animals can be confined in 2D or even immobilized, and liquids can be exchanged rapidly. These approaches enable advanced functional assays such as monitoring brain activity [15,16] or performing high-resolution behavior. [17] However, most of the highthroughput screening techniques are end-point assays based on fluorescence images [18,19] and do not translate well for phenotyping based on functional imaging. Single-phase microfluidic systems, although powerful, have limitations in the number of animals they can handle [17,[20][21][22][23] and the ability to modulate the experimental conditions at will. [16,[23][24][25] Multiphase microfluidics has unique attributes that make it well suited to tackle these challenges. These include the ability to partition, create independent containers of various sizes, generate precise chemical environments, perform serial processing, and consume ultra-low amounts of reagents. Multiphase microfluidics has led to advanced screening technologies impacting a broad range of disciplines, from particle synthesis [26,27] and synthetic biology, [28,29] to drug discovery, [30,31] and research in fundamental biology. [32,33] It has been applied successfully for numerous single-cell screens [34] and is gaining popularity for multicellular organisms such as spheroids and organoids. [35][36][37][38] In contrast, the development of multiphase microfluidics techniques for small animals [39][40][41][42][43] has seen a slow progress because of several technical bottlenecks. For instance, passive encapsulation techniques [44] are not adaptable to motile, multi-cellular organisms, mining the throughput. Second, techniques for controlling the droplet composition post-production rely mainly on merging, [45] which limits the ability to dynamically alter the droplet content. Furthermore, advanced active droplet manipulation tools [46][47][48][49] are inadequate as mechanical, thermal, and electrical-induced forces may create undesired stimulation. [50][51][52] Addressing these limitations is critical to fully exploit the potential of multiphase microfluidics as smallanimal screening technology.Here we describe a multiphase microfluidic platform to screen dynamic phenotypes in small animals. We demonstrate the use of this platform with the nematode C. elegans, Screening functional phenotypes in small animals is important for genetics and drug discovery. Multiphase microfluidics has great potential for enhancing throughput but has been hampered by inefficient animal encapsulation and limited control over the animal's environment in droplets. Here, a highly efficient single-animal encapsulation unit, a liquid exchanger system for controlling the droplet chemical environment dynami...

Advances in Microfluidics: Technical Innovations and Applications in Diagnostics and Therapeutics

“…Copyright 2022 Wiley (). (B) Liquid exchanger to exchange the chemical environment of one droplet with another without losing the encapsulated object (ref ). Adapted with permission from Automated and Dynamic Control of Chemical Content in Droplets for Scalable Screens of Small Animals, Aubry, G.; Milisavljevic, M.; Lu, H. Small , Vol.…”

“…Up to 88% of the original solution in the droplet could be exchanged with the new solution while keeping cell loss to less than 5%. Aubry et al developed a liquid exchanger passive system to dynamically control the chemical environment of small animals encapsulated in droplets (Figure B) . By careful use of interfacial forces, the liquid exchanger unit allows for adding and removing chemicals from a droplet and, therefore, generating chemical gradients inaccessible in previous multiphase systems.…”

“…The development of microfluidic screening platforms dedicated to small animals has paved the way for large-scale screens of ever-more advanced phenotypes. An automated droplet-on-demand platform with dynamic control over the droplet content can serve for the functional phenotyping of small animals . A microfluidic pipeline for large-scale smFISH imaging of C. elegans embryos gives the means to investigate temporal variations of gene expression in early embryogenesis. , An all-glass device compatible with whole-brain in vivo imaging using light-sheet microscopy can provide insights into zebrafish brain function with high spatiotemporal resolution …”

Daphnia as a model organism to probe biological responses to nanomaterials—from individual to population effects via adverse outcome pathways

“…For multicellular organisms, some ecotoxicity studies were reported, for example, with: rotifers (Brachionus calyciflorus) (Cartlidge and Wlodkowic, 2018), Crustacea (Artemia sp.) (Huang et al, 2015), Allorchestes copressa (Cartlidge et al, 2017) and Daphnia magna (Huang et al, 2017;Tabatabaei Anaraki et al, 2018), nematode (Caenorhabditis elegans) Zhang et al, 2021;Aubry et al, 2022) and fish (Danio rerio) models (Yang et al, 2016;Khalili and Rezai, 2019).…”

Worm Generator: A System for High-Throughput in Vivo Screening

“…As one of the best-investigated small animal models, Caenorhabditis elegans ( C. elegans ) has been extensively employed in large-scale genetic and pharmacological screenings, benefiting from its various experimental advantages including tiny size, body transparency, short life span, well-characterized genetics, and low-cost maintenance. − Moreover, numerous C. elegans disease models have also been well developed to recapitulate distinctive phenotypes of human diseases, such as pathogen infections, which has paved the way for deep profiling candidate therapeutics at a whole organism level. − However, it is still challenging to screen C. elegans for behaviors that unfold over a long period and evaluate quickly and accurately in real time via traditional manually assisted screening techniques. Even though various microfluidic systems have been developed to screen C. elegans , the complex external systems have highly restricted their applicability. − Particularly, almost all the proposed platforms are based on microscopic imaging with a high-speed camera, which further limited their employment due to the high cost and low throughput in long-term evaluations. Therefore, a practical, cost-effective and high-throughput system for in vivo screening of C. elegans is still clearly needed in the fields of biology and pharmacology.…”