One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells.

Kerstin Göpfrich,Oskar Staufer,Barbara Haller,Ilia Platzman,Yannik Dreher,Ulrike Mersdorf,Joachim P Spatz

doi:10.1021/acssynbio.9b00034

Kerstin Göpfrich, Oskar Staufer + Show 5 more

Open Access

https://doi.org/10.1021/acssynbio.9b00034

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Abstract

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil–surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

Highlights

Lipid bilayer membranes define the boundaries of virtually all living cells
We obtain a giant unilamellar vesicles (GUVs) inside the water-in-oil droplet, which we will refer to as “droplet-stabilized GUV”
Detailed step-by-step instructions with tips for troubleshooting and a video protocol of the entire GUV formation process are provided in the Supporting Information (Text S1, Video S1)

Summary

Introduction

Lipid bilayer membranes define the boundaries of virtually all living cells. The creation of artificial phospholipid vesicles gave insights into the biophysical properties of cellular membranes and led to the development of new drug delivery systems.[1]. Alternatives have been proposed, including solvent evaporation,[11] osmotic shock,[1] gel-assisted swelling,[7] and inverted emulsions.[12] A good overview of these techniques and potential artifacts can be found elsewhere.[13,14] Notably, the water-in-oil emulsion transfer method[15] greatly improved the encapsulation efficiency, which led to the successful reconstitution of protein expression systems in GUVs.[16] Recently, different methods for the microfluidic formation of GUVs were demonstrated.[17−22] These methods received increased attention for the bottom-up assembly of synthetic cells, as they feature high-yield and homogeneous size, and most importantly, encapsulation of biomolecules via the aqueous inlet is straightforward.[23] establishing microfluidic technologies may be timeconsuming and, in the case of PDMS-based microfluidics, requires clean room facilities. The GUV formation method features a Letter straightforward reconstitution of membrane proteins and formation of multicompartment systems as well as high encapsulation efficiency

Methods

Results

Conclusion