Sedimentation-based confinement of individual Giant Unilamellar Vesicles in microchamber arrays with a dynamically exchangeable outer medium

Giant unilamellar vesicles (GUVs) are an ideal model to study cellular membrane functions in vitro, yet difficult to manipulate due to their fragile nature, especially when subjected to dynamic change of their external microenvironment. Here, we introduce an original microfluidic concept for constrain-free confinement of individual GUVs in microchambers with a dynamically exchangeable outer medium. With this method, GUVs self-confine in an array of laterally separated microchambers by sedimentation, avoiding any mechanical constrain and membrane deformation while allowing time-resolved microscopy observation. A microfluidic channel above the chambers allows a diffusion-based exchange of the GUV outer medium that can be completed in a few seconds for fast-diffusing molecules to about one minute for large proteins in a viscous medium. We numerically establish the geometric and flow parameters optimizing medium exchange while preventing GUV from lifting out. We experimentally demonstrate that different aqueous solutions separated by air plugs can be flowed into the channel by taking advantage of a polydimethylsiloxane-based hydrophilic channel wall. We also exploit the possibility to manipulate microliter sample volumes and dynamically control the external environment of GUV for in situ observation of membrane binding protein cell-free expression. We find in particular that the membrane-targeting sequence of Bacillus subtilis MinD binds to GUVs and induces extensive membrane tubulation. This technically simple method offers a robust way to confine GUVs and dynamically control their outer medium, thus constituting an ideal platform to study the spatio-temporal response of reconstituted membranes and/or synthetic cell studies subjected to dynamic micro-environments.