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.

We have developed a technology to manufacture multilayered lipid vesicles. Individual bilayers are deposited layer‐by‐layer, allowing us to regulate the number of bilayers and their individual composition. This depiction of our structures portrays the self‐assembly underpinning our method (bottom left) and our discovery that phase‐separated domains align with one another through the layers (yellow). The undulating membranes represent the fluctuation analysis technique that we use to show that the mechanical properties of the vesicle scale with the number of layers. More information can be found in the Full Paper by Y. Elani et al. Image by Zehua Hu.