University of Cambridge: Giant unilamellar vesicle production and testing
About The Keyser lab
The Keyser lab is a group of researchers at the Cavendish Laboratory, University of Cambridge, UK. Since it was founded in 1874, the Cavendish Laboratory has been at the forefront of discovery in physics, with the core to be experimental physics supported by excellence in theory. The department promotes world-leading experimental and theoretical physics in all its diversity. Scientists from the Keyser lab study the physics of ions, macromolecules and particles in confined geometries at the single molecule/-particle level is of particular interest. To exert maximum control over all parameters for their experiments, they make use of several cutting-edge techniques such as DNA self-assembly (origami), optical trapping, electrophysiology, and microfluidics and nanofluidics.
The team combines researchers with expertise in physics, engineering, physical chemistry, biochemistry/biology, and micro- and nanofabrication.
“Microfluidics presents various advantages to researchers who need small volumes and high throughput in answering their scientific questions. In our lab, we use microfluidic devices for standardization and control of experimental parameters like concentration and timing. In the complex (biological) systems we are working on, the mentioned characteristics are fundamental in collecting reliable meaningful statistics, and microfluidics in combination with light microscopy offers just that. We also heavily rely on the ability to rapidly prototype devices, as we can design bespoke solutions at minimal production cost and time.”
“We use the pressure-based pumps from Fluigent for experiments that require swift responsiveness when manipulating fluids, and fine tuning at low flow rates. We use the Fluigent systems during fabrication and running of the microfluidic chips. The ability to pump in air at high precision makes the Fluigent pressure-based systems ideally suited to selectively coat and functionalize micro-channels within a microfluidic network. After coating we then fill the devices with the experimental solutions and use the pressure controls to move fluids around, open and close valves and carefully time the introduction of small molecules in the experiments.“
Kareem Al Nahas, University of Cambridge
Giant Unilamellar Vesicles (GUVs) production and testing using the octanol-assisted liposome assembly (OLA) method
Introduction
Liposomes of several microns in size, referred to as giant unilamellar vesicles (GUVs), are routinely used in research and drug screening including studies on drug permeation and transport through membrane pores, lipid scrambling and membrane fluctuation1. They offer the advantages of having well-defined lipid compositions, being easy to image, and being controlled systems for studying transport processes2. In 2016, the Dekker laboratory from the Delft University of Technology developed a novel microfluidics-based method: Octanol-Assisted Liposome Assembly (OLA)3, which allows to generate GUVs with higher monodispersity and a greater control of sizes compared to traditional methods, and with a faster process compared to alternative microfluidic methods.
Scientists from the University of Cambridge integrated the OLA technique into their microfluidic platforms (“lab on a chip” devices) for quantifying drug permeation and antibiotic efficacy on biomimetic vesicle membranes1,2. In a research paper published in May 2020, scientists from the University of Cambridge and Exeter produced GUVs with tunable binary lipid mixtures to determine lipid diffusion in OLA liposomes. Below, we summarize the GUVs production process from the last article.
In these studies, and the one following, Fluigent pressure-driven flow controllers were used to generate GUVs with the OLA method.
Fluigent pressure-based flow controllers for monodisperse, stable, and controlled vesicle formation
The microfluidic chip for OLA consisted of three inlets for the inner (IA) and outer aqueous (OA) phases, and the lipid-octanol (LO) phases (figure below). The vesicles are formed at a six-way junction and flow through the channel outlet where they can be recovered4.
For the production of GUVs, fluids were controlled using Fluigent pressure-based flow controllers. The authors typically operated the chip with input pressures of 40 mbar for the IA and LO phases and 100 mbar for the OA phase. The total flow rate was estimated to be on the order of 10 µL/h. For precise flow measurements, a Fluigent Flow Unit can be added on the fluidic path. OLA microfluidic technique combined with pressure control also allows one to adjust the sizes of the generated vesicles by adjusting the microfluidic pressures of the IA, LO, or OA phases. Such a degree of control is difficult to achieve using standard methods such as electroformation.
References
- Al Nahas, K. et al. A microfluidic platform for the characterization of membrane active antimicrobials. Lab Chip 19, 837–844 (2019).
- Schaich, M. et al. An Integrated Microfluidic Platform for Quantifying Drug Permeation across Biomimetic Vesicle Membranes. Mol. Pharm. 16, 2494–2501 (2019).
- Deshpande, S., Caspi, Y., Meijering, A. E. C. & Dekker, C. Octanol-assisted liposome assembly on-chip. Nat. Commun. 7, 1–9 (2016).
- Schaich, M., Sobota, D., Sleath, H., Cama, J. & Keyser, U. F. Characterization of lipid composition and diffusivity in OLA generated vesicles. BBA – Biomembr. (2020) doi:10.1016/j.bbamem.2020.183359.
More information
- Keyser lab website: http://people.bss.phy.cam.ac.uk/~ufk20/
- Keyser lab website: http://people.bss.phy.cam.ac.uk/~ufk20/
- Cambridge Cavendish Laboratory website: https://www.phy.cam.ac.uk/
