Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems

Abstract

In this manuscript, we introduce a simple, off-the-shelf approach for the on-demand creation of giant unilamellar vesicles (GUVs) or multicompartment synthetic cell model systems in a high-throughput manner. To achieve this, we use microfluidics to encapsulate small unilamellar vesicles in block-copolymer surfactant-stabilized water-in-oil droplets. By tuning the charge of the inner droplet interface, adsorption of lipids can be either inhibited, leading to multicompartment systems, or induced, leading to the formation of droplet-stabilized GUVs. To control the charge density, we formed droplets using different molar ratios of an uncharged PEG-based fluorosurfactant and a negatively-charged PFPE carboxylic acid fluorosurfactant (Krytox). We systematically studied the transition from a multicompartment system to 3D-supported lipid bilayers as a function of lipid charge and Krytox concentration using confocal fluorescence microscopy, cryo-scanning electron microscopy and interfacial tension measurements. Moreover, we demonstrate a simple method to release GUVs from the surfactant shell and the oil phase into a physiological buffer -providing a remarkably high-yield approach for GUV formation. This widely applicable microfluidics-based technology will increase the scope of GUVs as adaptable cell-like compartments in bottom-up synthetic biology applications and beyond.

Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review

“…Previously, posts or hurdles had been implemented to trap biological cells [47] or droplets, [48][49][50] but here they were combined with pressure actuated valves and used to trap single GUVs. [49,50,[52][53][54] The addition of integrated valves allows rapid and homogeneous fluid exchange of a particular subset of trapped vesicles in less than 0.5 s enabling multiple unique experiments per device.…”

“…In the context of using the bottom-up approach for the construction artificial cells, there are a number of functionalities currently being explored. [53,107] [105,106] Giant vesicles, and in particular GUVs, are excellent compartments systems well suited for the reconstitution of the necessary machinery to achieve these goals and microfluidic technologies for handling them will play a major role.…”

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

“…C) Charge‐controlled microfluidic for formation of multicompartmental vesicles. Reproduced with permission . Copyright 2018, Royal Society of Chemistry.…”

Compartments for Synthetic Cells: Osmotically Assisted Separation of Oil from Double Emulsions in a Microfluidic Chip

“…[1][2][3] In parallel, they attract increasing attention as cell-like compartments in bottom-up synthetic biology,i n which the long-term goal is to build am inimal cell from scratch. [18] In recent years, severalo ther,c oncep-tually similar,m ethods have been developed, with the aim of providing higher productivity andb etter control, namely,m icrofluidic jetting, [19] continuous droplet interface crossing encapsulation (cDICE), [20] microfluidic formation of droplet-stabilized vesicles, [21] and microfluidic production of w/o/w double emulsions. [2] Conventionalm ethods for the productiono fl iposomes comprise gentle hydration, [9,10] swelling on polymer cushions, [11,12] and electroformation.…”

“…[13,14] These methods are not alwayso ptimal due to the low GUV yield in physiological buffer;p oor encapsulation efficiency; [2,15,16] and, in some cases, harsh conditions to which delicate biomolecules and smaller vesicles are exposed during preparation. [18] In recent years, severalo ther,c oncep-tually similar,m ethods have been developed, with the aim of providing higher productivity andb etter control, namely,m icrofluidic jetting, [19] continuous droplet interface crossing encapsulation (cDICE), [20] microfluidic formation of droplet-stabilized vesicles, [21] and microfluidic production of w/o/w double emulsions. [18] In recent years, severalo ther,c oncep-tually similar,m ethods have been developed, with the aim of providing higher productivity andb etter control, namely,m icrofluidic jetting, [19] continuous droplet interface crossing encapsulation (cDICE), [20] microfluidic formation of droplet-stabilized vesicles, [21] and microfluidic production of w/o/w double emulsions.…”