Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

Abstract

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

Keywords: artificial cells; biomimetics; droplet interface bilayers; microfluidics; synthetic biology; vesicles.

Figure 1. Schematic of a hypothetical vesicle-based artificial cell which contains some key cellular components and features

(i) Membrane of defined biomolecular composition and asymmetry. (ii) Dynamic cell-free expression of proteins by IVTT using rudimentary genetic circuits. (iii) Incorporation of non-biological components. (iv) Communication between an artificial cell and a biological cell via an engineered signalling cascade. (v) Embedded responsive protein pores that open/close according to external stimuli. (vi) Membrane-embedded recognition modules (e.g. antibodies). (vii) Sub-compartmentalization inside cells into regions with distinct chemical environments for multi-step reactions.

Figure 2. Strategies for microfluidic generation of vesicles

(A) Droplets are formed on-chip, and then expelled above an oil–water column. Lipid is dissolved in the oil phase, and an interfacial monolayer assembles around the droplet and at the water/oil interface of the column. The droplets are loaded with sucrose, and therefore descend through the column under gravity. As droplets enter the lower water phase they are encased in a second interfacial monolayer, resulting in a bilayer membrane. (B) Schematic of a device where both droplet generation, and subsequent conversion into vesicles, occurs on chip, with the aid of a triangular microfabricated post, guiding droplets across the phase boundary. (C) Vesicle generation from double emulsions formed with a microfluidic device. The emulsions are stabilized by lipids and, as the intermediate oil phase is extracted into the external phase, vesicles are generated.

Figure 3. Higher-order membrane assemblies

(A) Schematic of a three-compartment vesicle-based cell constructed from droplet precursors, where each compartment exists in a distinct biochemical environment, and is engineered to perform one step of a multi-step enzymatic reaction. The reaction intermediates move between compartments via transmembrane pores. (B) Schematic of a bespoke droplet printer capable of printing thousands of lipid-coated droplets, leading to a tissue-like material. (C) This artificial tissue can be functionalized to exhibit collective properties, including self-folding and transmittance of electrical signals down pre-defined paths through transmembrane proteins. Panels B and C were modified from [46]: Villar, G., Graham, A.D. and Bayley, H. (2013) A tissue-like printed material. Science 340, 48–52, reprinted with permission from AAAS.