A Refined cDICE Platform for the Controlled Production of Lipid Giant Unilamellar Vesicles

Continuous Droplet Interface Crossing Encapsulation (cDICE) is well known to be an effective alternative to the manual reverse emulsion method to produce giant unilamellar vesicles (GUV). It provides high encapsulation efficiency and enhanced user control over process variables, comparable to that offered by microfluidic approaches, without involving extensive infrastructure requirements such as clean rooms or lithography. The cDICE method can be challenging to implement because its performance depends on the precise configuration of the experimental setup and careful adherence to protocol details that are easily overlooked. The aim of this paper is to provide a practical, comprehensive, step-by-step guide to operating an improved cDICE system, covering the design of the rotating chamber through to the extraction of the resulting GUVs. This paper will also quantify the influence of ambient humidity, an often-mentioned factor in vesicle generation, and compare small molecule encapsulation efficiency and vesicle size characteristics with the manual reverse emulsion phase transfer method. The often overlooked influence of sonicator choice for lipid oil solution preparation will be examined, highlighting the downstream effects that the pre-cDICE preparation steps can have on the overall effectiveness of the vesicle generation technique demonstrate the applicability of the improved cDICE set up to synthetic-cell research, a protocol is presented for producing GUVs, containing a cell-free protein synthesis system that expresses eGFP. GUVs containing cell-free expression systems are widely used as model synthetic cells because they provide a membrane-bound compartment in which genetic constructs can be expressed and studied under controlled conditions.