Picodroplet microfluidics - specialist chemicals for single cell analysis

Picodroplet microfluidics is rapidly becoming the technology of choice for high-throughput single cell analysis, providing an easy-to-use tool to isolate single cells in picolitre-volume, water-in-oil droplets (picodroplets), stabilised by a biocompatible surfactant layer. The use of an appropriate surfactant, that can mirror the function of phospholipid membranes in biological systems, is essential for efficient and robust picodroplet manipulation. This article explores novel multifunctional and biocompatible surfactants that create a uniquely protective environment to support cell viability and integrity during incubation and shield cells against shear stress during subsequent analysis.

Picodroplet technology

Microfluidic systems based on picodroplet technology provide a high-throughput and sensitive method for performing quantitative screening assays at rates unachievable in conventional laboratory workflows [1]. By encapsulating single cells in picolitre volume compartments, each picodroplet acts as a miniature ‘test tube’ in which single cells can be stored, incubated, and sorted for target events, then dispensed for further analysis. This picodroplet-based approach offers flexibility in assay design, enabling a broad spectrum of applications, including secreted proteins, isolating rare cells from large populations, improving yields of therapeutic antibodies, and undertaking genome-editing analysis at the single cell level.

The range of applications for picodroplet technology has expanded as a result of improvements in surfactants. Surfactants maintain the stability of emulsions in different experimental conditions and for extended periods of time. When generating a picodroplet, the surfactant molecules rapidly arrange themselves on the surface of the picodroplet, creating a layer that stabilises the water and oil interface and reduces surface tension [2]. The steric repulsion between the water and oil layers, facilitated by surfactant molecules, prevents aqueous picodroplets from fusing or breaking during analysis.

Figure 1. Depiction of picodroplets generated using comparable commercially available surfactants, following high temperature treatment up to 95°C.

Multifunctional biocompatible fluorosurfactants

While ionic biocompatible surfactants are available, they are generally considered to perform poorly relative to non-ionic versions. So, to provide the inert and biocompatible water-oil interface that allows enhanced gas exchange during effective single cell analysis, the surfactant must be non-ionic. The selected surfactant must also maintain picodroplet stability at various temperatures and throughout the multi-step experimental workflows during which picodroplets are stored, reinjected, incubated and undergo various other manipulations. Novel multifunctional and biocompatible fluorosurfactants have been developed and commercialised to meet the demands of high-throughput single cell analysis. By emulating the function of biological phospholipid membranes specialist surfactants, composed of a unique molecular structure and robust fluorophobic moiety/hydrophilic moiety ratio, can offer several advantages [2]. Benefits include batch-to-batch reproducibility, picodroplet stability, consistent reliability, stability at various temperatures, and functionality at low voltage for sorting. This last facilitates a gentler method for analysing picodroplets and supports cell viability over time. These advantages enable new and improved applications across single cell analysis, cell and molecular biology assays, gene transfection, cell growth studies, electrospray ionisation mass spectrometry, double emulsions, and hydrogel microfluidics [1-8].

Performance enhancements in multifunctional biocompatible fluorosurfactants can be demonstrated by investigating the lowest end point interfacial tension (IFT) and Critical Micelle Concentration of comparable surfactants. Chemicals with the lowest values support greater picodroplet stability, compared to others available. Confirmation of the robust and stable picodroplets generated following 95°C temperature treatments can be demonstrated through imaging (Figure 1), the surfactants showing low levels of picodroplet fusion offering superior performance. 

Given the superior performance characteristics of novel biocompatible surfactants, researchers can use picodroplets as robust microreactors [2]. Cells are protected in a cell-friendly, microscale environment able to support cell viability and integrity while undergoing various manipulations [1]. The surfactant promotes high gas exchange rates to maximise cell viability and generates picodroplets that are stable for many days. In studies that demonstrate cells in surfactant-stabilised picodroplets can indeed be cultured for prolonged periods, researchers also show that (depending on the cell line), when an appropriate surfactant is used cells can be tested and then seeded for clone outgrowth up to 72 hours after retrieval (Figure 2) [5].

Author: Olivia Hughes is Digital Marketing Associate for Sphere Fluidics, spherefluidics.com

References:

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[5] Shvets E, Lui X, Hughes O and Craig F. Cell Engineering in Picodroplets (2021). https://spherefluidics.com/resources/application-materials/ Accessed July 30, 2021.

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