Manufacture of Multilayered Artificial Cell Membranes through Sequential Bilayer Deposition on Emulsion Templates

Abstract

Efforts to manufacture artificial cells that replicate the architectures, processes and behaviours of biological cells are rapidly increasing. Perhaps the most commonly reconstructed cellular structure is the membrane, through the use of unilamellar vesicles as models. However, many cellular membranes, including bacterial double membranes, nuclear envelopes, and organelle membranes, are multilamellar. Due to a lack of technologies available for their controlled construction, multilayered membranes are not part of the repertoire of cell‐mimetic motifs used in bottom‐up synthetic biology. To address this, we developed emulsion‐based technologies that allow cell‐sized multilayered vesicles to be produced layer‐by‐layer, with compositional control over each layer, thus enabling studies that would otherwise remain inaccessible. We discovered that bending rigidities scale with the number of layers and demonstrate inter‐bilayer registration between coexisting liquid–liquid domains. These technologies will contribute to the exploitation of multilayered membrane structures, paving the way for incorporating protein complexes that span multiple bilayers.

Chemical Communication in Artificial Cells: Basic Concepts, Design and Challenges

“…The idea of engineering synthetic organelles to co-localize the components of an engineered metabolism has gained considerable interest in recent years. Reproducing such complex pathways in artificial cells can be achieved by building multi-compartmentalized ensembles ( Lee et al, 2018 ; Belluati et al, 2020 ; Ip et al, 2021 ; Wen et al, 2021 ). In a recent example, Robinson et al developed a microfluidic platform to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic mimics of eukaryotic cells.…”

Layer-by-layer assembly of multi-layered droplet interface bilayers (multi-DIBs)

“…Trilayer droplets are produced by depositing a further lipid monolayer on the outer surface of a giant vesicle through a modification of an emulsion-based lipid templating approach previously used to manufacture multilamellar vesicles. 32 The presence of multiple lipid layers around the droplet was confirmed through a fluorescent quenching assay before stochastic collisional based contact induced by centrifugation was achieved to produce the multi-layered DIBs, which we term multi-DIBs. The properties of the multi-DIBs were then explored through biophysical measurements and fluorescence-based assays.…”

Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells

“…While this method does offer separate control over the inner and outer aqueous compartments and their membranes, it can suffer from low or inhomogeneous encapsulation efficiencies. Alternatively, instead of encapsulating inner compartments within liposomes, a layer-by-layer technique can be employed to form multicompartment structures that mimic complex cellular architectures . However, this method results in a polydisperse size distribution and has limited space in between the compartments to perform reactions.…”

“…Alternatively, instead of encapsulating inner compartments within liposomes, a layer-by-layer technique can be employed to form multicompartment structures that mimic complex cellular architectures. 17 However, this method results in a polydisperse size distribution and has limited space in between the compartments to perform reactions. Successful chemical communication has been demonstrated within multicompartment lipidbased structures by the permeation of trigger molecules 18 and by using a temperature-controlled release of substrate molecules from the inner compartments.…”