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 molecular processes of living systems is to take the opposite approach of reconstituting cellular behaviors using a minimal set of biological components. A critical part of this approach involves the reliable encapsulation of biomolecules in a confined volume, a key feature of a cell.

Several strategies exist for encapsulating biomolecules for studying biomimetic systems. The most biologically relevant system is lipid bilayer membranes, which mimic the biochemical and physical constraints imposed by the cell's plasma membrane. Formation of giant unilamellar vesicles (GUVs) by electroformation⁷, one of the most widely used techniques for GUV generation¹⁴, typically has a poor encapsulation yield due to its incompatibility with high salt buffer⁸. Electroformation also requires large sample volumes (>100 μl), which could be a problem for working with purified proteins, and inefficiently incorporates large molecules due to difficulty of diffusion between closely spaced lipid layers. Several microfluidic approaches for generating lipid vesicles have been developed. The double emulsion methods, which pass components through two interfaces between layers water-oil-water (W/O/W), relies on the evaporation of a volatile solvent to drive lipid bilayer formation⁹. Others have used a microfluidic assembly line that produces a continuous stream of lipid bilayer vesicles¹⁰ or in two independent steps¹¹. We have developed an alternate technique based on rapidly applying fluid pulses against a droplet interface bilayer¹² to produce GUVs of controlled size, composition, and encapsulation. Our approach, known as microfluidic jetting, offers the combined advantages from several existing vesicle generation techniques, providing an approach for creating functional biomolecular systems for investigating a variety of biological problems.

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 scissors or a laser cutter to serve as a bottom to the wells.
3. Apply a thin but complete layer of quick-drying adhesive to one side of the 0.2 mm acrylic and glue it to the bottom of the chamber. Hold the 0.2 mm acrylic tightly in place against the bottom of the chamber and dispense the glue at the interface to allow the glue to create a seal but avoid covering the viewing area. Be sure to align the acrylic so that its edge is flush with the edge of the chamber wall and it completely covers the infinity chamber cutout. This will allow for sufficient jet penetration and prevent leakage of the well.
4. Cut two small pieces of natural rubber to cover the drilled holes. Apply quick-drying adhesive around the hole. Place the rubber over the hole and press on all areas with a pair of forceps to secure. Repeat for both drilled holes. Be sure that all glued connections are complete seals so as to prevent any leakage.
5. Using a 23 G, 1 in needle, poke a hole in the natural rubber on both sides of the chamber to facilitate insertion of the piezoelectric inkjet tip.

2. Experimental Preparation

Store stock lipid solution in chloroform in a -20 °C freezer. For this study, either 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was used. 2 ml of 25 mg/ml lipid solution is typically prepared in this protocol and will last for two months when stored at -20 °C.

1. To resuspend the lipid in n-decane, first transfer it from a stock vial to a small glass jar, and gently dry under argon or nitrogen. Hold the jar at an angle while drying to expose more surface area to the gas, allowing the lipid to dry faster.
2. With the cap of the jar only slightly screwed on, allow the dried solution to sit under vacuum in a desiccator for 1-2 hr. Then add n-decane to resolubilize the lipid at 25 mg/ml. Vortex the lipid solution briefly and sonicate it in a bath sonicator for 15 min. Store the reconstituted lipid in n-decane at -20 °C.
3. Prepare a stock sucrose solution. In a 1.5-ml microcentrifuge tube, add 900 µl of 300 mM sucrose and 100 µl of 1% methylcellulose (MC). Methylcellulose is added to increase viscosity of the solution, which aids in jetting. Optional step: Add an optional 1 µl of dark-colored food dye or fluorescent beads to lend more contrast or fluorescence in imaging, respectively. This solution is a 300 mOsm sucrose solution designed to match cellular osmolarity and to provide contrast during microjetting.
4. Draw the solution into a disposable 1 ml plastic syringe. While holding the syringe with the tip facing upwards, flick the shaft repeatedly to expel any bubbles toward the tip, and push the plunger to eject the trapped air. Be sure to evacuate all air from the syringe before proceeding, as it will interfere with proper piezoelectric contraction responsible for jetting.
5. Install a 0.22 µm filter on the end of the syringe. To prevent air from being trapped in the filter, hold the syringe vertically and push the plunger until a droplet is formed above the tip. Note: A 33 mm diameter syringe filter unit was found to work best, but alternative filters as small as a 3 mm diameter syringe filter can be used to reduce dead volume.
6. Unscrew the female Luer adapter of the inkjet, and securely press it in place over the end of the filter. Again, eject fluid to prevent trapping air. Screw in the top of the inkjet. Fluid should travel to the tip of the inkjet after it is completely attached.

3. Readying the Equipment

1. Using a v-clamp, mount the syringe assembly on the microscope stage. Attach the wire from the inkjet to the inkjet controller. Note: A custom stage was built for this protocol; while the stage design can be determined by the user, it is critical to have independent x-y-z control of the syringe and x-y control of the sample holder.
2. Determine the magnification and necessary lens combination to achieve the desired imaging. Here a 10X objective and 10X eyepiece are used.
3. Use a high-speed camera (≥1,000 fps) to visualize the jetting and vesicle generation. Prior to imaging, perform necessary camera calibration. Utilize image-based auto-trigger within the camera software to initiate image capture.
4. Mount the infinity chamber onto the microscope stage. Secure the chamber by taping it into place on the stage.
5. Carefully align the inkjet tip with the hole punctured in the natural rubber (see Figure 1b). To do this, bring the inkjet close to the chamber and adjust the positioning by eye, then make more precise adjustments through the microscope lens. Proceed cautiously, as coarse movements can damage the inkjet tip.
6. Once the inkjet is aligned, back the syringe assembly away from the chamber to prevent any damage to the inkjet during loading of the wells. Be sure that motion of the inkjet is unidirectional so that it remains aligned with the hole in the membrane.
7. Press gently on the plunger of the syringe assembly until a small droplet forms at the inkjet nozzle. This will provide some initial backpressure.
8. Input the jetting parameters. For a trapezoidal bipolar wave, use the following parameters: 20 kHz pulse frequency, 3 µsec rise time, 35 µsec pulse duration, 3 µsec fall time, 65 V applied voltage (pulse amplitude), and 100 jet pulses per trigger (pulse number). However, the applied voltage (pulse amplitude) and pulse number (jet pulses per trigger) can be varied to control vesicle size.

4. Vesicle Generation

1. Add 25-30 µl lipid solution suspended in n-decane to the infinity chamber, covering the full surface of both wells.
2. Add 25 µl glucose (of same osmolarity as the sucrose solution) to the outermost edge of one well, pipetting slowly and smoothly. Upon deposition, a drop of glucose should form, because the glucose and lipid solution do not mix. Add another 25 µl of glucose to the opposite well, which will make another drop and form the lipid bilayer membrane in the middle of the chamber within 5-10 min.
3. Insert the inkjet through the natural rubber, and carefully guide it towards the droplet interface bilayer. Approach the bilayer slowly, as the introduced inkjet will displace volume and can rupture the bilayer.
4. When the inkjet is within ~200 µm, apply the jetting with the settings described in step 3.8. The distance from the bilayer may vary primarily depending on the voltage and pulse number, among other parameters. This protocol recommends slowly increasing settings (voltage and pulse number) and observing bilayer deformation.

5. Cleaning the Equipment

1. Detach the syringe assembly from the microscope stage, and dispose of the 1 ml plastic syringe and filter.
2. To clean the inkjet, aspirate the following solutions in order by dipping the tip in the solution 7-10x each: 70% ethanol, 2% Neutrad solution in warm water, 70% ethanol, and ddH₂O. If the inkjet doesn't fit securely on the aspiration pipette, cut a pipette tip to form an adapter.
3. Dry the chamber with tissue. Place the infinity chamber in a 250 ml beaker with 2% v/v Neutrad in warm water, and sonicate for 5-10 min. After sonication, thoroughly dry the wells under compressed air. Any moisture in the wells can compromise the stability of the lipid bilayer membrane, so it is also recommended that the chambers are placed in an oven at 60 °C for 15 min.

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

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