Multivesicular droplets: a cell model system to study compartmentalised biochemical reactions

Abstract

Multivesicular vesicles (MVVs) are artificial liposomal structures widely used as a platform to study the compartmentalisation of cells and as a scaffold for artificial cell/protocell models. Current preparation techniques for MVVs, however, offer poor control on the size, lamellarity, and loading of inner lipid vesicles. Here, we introduce a microfluidic device for the production of multivesicular droplets (MVDs): a novel model system combining the ease of use and control of droplet microfluidics with the biological relevance of MVVs. We use a perfluorinated carrier phase with a biocompatible surfactant to generate monodisperse droplets of an aqueous giant unilamellar lipid vesicle suspension. The successful on-chip formation and stability of MVDs is verified through high-speed microscopy. For bright field or fluorescence microscopy inspection, the MVDs are trapped in an array where the integrity of both lipid vesicles and droplets is preserved for up to 15 minutes. Finally, we show a two-step enzymatic reaction that takes place across the lipid vesicle membranes; the second reaction step occurs in the vesicle's interior, where the enzyme is encapsulated, while both the substrate and fluorescent product permeate across the membrane. Our approach opens the possibility to mimic artificial organelles with optimised reaction parameters (pH, ions, etc.) in each compartment.

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

“…What is more, controllable fabrication of two adjoining lipid bilayers can be realized by the compartmentalization, which would be contributable to implement other functions of cells. Dittrich and co‐workers also reported the multivesicular droplets derived cell model for the study of compartmentalized biochemical reactions …”

“…Dittrich and co-workers also reported the multivesicular droplets derived cell model for the study of compartmentalized biochemical reactions. [215] Cells are containing a dazzling variety of morphologies, such as rods, spheres, spirals, triangular, flattened square shapes and so on. Although cell morphology is important for cellular dynamics, the current artificial cells studies mainly use spherical models, because of the lack of methods to manipulate artificial cell shape.…”

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications

“…The most demonstrated and utilized enzymatic cascade is the glucose oxidase (GOx) and horseradish peroxidase (HRP) cascade, in which GOx oxidizes glucose to produce H 2 O 2 , a oxidizing substrate for HRP. [39,131,150,233,247,259,[328][329][330][331][332][333][334][335] The GOx-HRP cascades can also be performed in coacervate droplets as membraneless organelles, by cationizing or anionizing GOx and HRP respectively (Figure 16a). [233,247,253,259,270,333,334,336,337] A complicated version of the glucose cascade is presented in a synthetic beta cell, a multicompartmental vesosomes composed of an outer vesicle with glucose transporter 2 (GLUT2) and inner pH-responsive vesicles containing insulin (Figure 16b).…”

Droplet microfluidics for the construction of compartmentalised model membranes

“…It should also be noted that recently Nuti et al demonstrated the microfluidic production of multi-vesicular droplets (vesicles in droplets), that retain the biological relevance of their vesosome analogues, yet are easier to use and control. 125 These were formed by encapsulating vesicles generated offchip into droplets using a FFJ, where encapsulation numbers followed a Poisson distribution. A compartmentalised two-step enzymatic cascade was demonstrated using enzymes present in the interior of the encapsulated vesicle 'organelles' and the external vesicle 'cytoplasm'.…”

“…Glass capillaries 110 10-60 μm Multiple co-flow glass capillaries and flow focusing Polymersomes-in-polymersomes Glass capillaries 111 50-300 μm Co-flow and flow focusing glass capillaries Multi-compartment vesicles Single-droplet phase transfer 8,9,117 200-700 μm Tubing with droplets above phase-transfer column Glass capillaries 68 20-200 μm Multiple sequential co-axial capillaries Vesicles-in-vesicles Glass capillaries 123 50 μm (inner vesicles) Multiple sequential co-axial capillaries 100 μm (outer vesicles) Vesicles-in-droplets Soft lithography 125 50 μm (droplets); 2-30 μm (vesicles)…”