Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Summary

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Abstract

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies^1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation³. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Introduction

It has become increasingly clear that cell biology is a multi-scale problem that involves integrating our understanding from molecules to cells. Consequently, knowing precisely how molecules work individually is not sufficient to understand complex cellular behaviors. This is partly due to the existence of emergent behaviors of multi-component systems, as exemplified by the reconstitution of actin network interaction with lipid bilayer vesicles⁴, mitotic spindle assembly in Xenopus extract⁵, and spatial dynamics of bacterial cell division machineries⁶. One way to complement the reductionist's approach of dissecting the mol....

Protocol

1. Infinity Chamber Fabrication

1. Design the infinity chamber (named for its shape) using a computer assisted design (CAD) software, and save the file such that it is compatible with a laser cutter. To create this shape, separate two circles of diameter 0.183 in by a center-to-center distance of 0.15 in. Cut the chamber from 1/8- 3/16 in clear acrylic with the laser cutter. The infinity shape facilitates droplet interface bilayer formation and stability.
2. Drill a 1/16 in hole through the edge of the acrylic chamber to the infinity-shaped well. Repeat on the opposite side. Cut a small rectangle from a 0.2 mm acrylic sheet with sci....

Results

We have assembled a microfluidic jetting setup on a conventional inverted fluorescence microscope with a custom stage assembled from machined parts and manual micrometers (Figure 1). Characterization of the inkjet provides insight into the vesicle generation process. Varying the distance between the inkjet nozzle and lipid bilayer affects the force applied to cause deformation of the membrane. Close proximity to the bilayer focuses the jet stream and prevents the membrane from dispersing energy away from.......

Discussion

Many techniques have been developed for vesicle generation, including electroformation, emulsion, and droplet generation^14-16. However, new experimental techniques are necessary to allow for the design of biological systems with growing similarity to living systems. Microfluidic methods in particular have offered an increased level of control governing membrane unilamellarity, monodispersity of size, and internal contents^17,18, bringing vesicle models closer to biology. Furthermore, characterization.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Piezoelectric Inkjet	MicroFab Technologies	MJ-AL-01-xxx	xxx denotes orifice diameter in microns
Jet Drive III Controller	MicroFab Technologies	CT-M3-02
High-speed camera	Vision Research	MiroEX2
DPhPC lipid in chloroform	Avanti	850356C	Ordered in small aliquots in vials
33 mm PVDF filters, 0.2 µm	Fisher Scientific	SLGV033RS
1 ml Syringes	Fisher Scientific	14823434
n-Decane	Acros Organics	111871000
Glucose	Acros Organics	410950010
Sucrose	Sigma-Aldrich	S7903-1KG
Methylcellulose	Fisher Scientific	NC9084958
1/8 in Acrylic	McMaster Carr	8560K239	CAD designs for the infinity-shaped chamber are available upon request
0.2 mm Acrylic	Astra Products	Clarex clear 001
Acrylic Cement	TAP Plastics	10693
Loctite 495 Superglue	Fisher Scientific	NC9011323
Loctite 3494 UV Strengthening Adhesive	Strobels Supply	30765
Natural rubber	McMaster Carr	85995K14
Custom stage	homemade	N/A	CAD designs are available upon request