We thank Mike Vahey from the Fletcher Lab at the University of California, Berkeley for advice on the microjetting parameters. This work was sponsored by NIH grant DP2 HL117748-01.

1. Infinity Chamber Fabrication
<ol>
	<li>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&#160;by a center-to-center distance of 0.15 in. Cut the chamber from 1/8- 3/16 in&#160;clear acrylic with the laser cutter. The infinity shape facilitates droplet interface bilayer formation and stability.</li>
	<li>Drill a 1/16 in&#160;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&#160;mm acrylic sheet with scissors or a laser cutter to serve as a bottom to the wells.</li>
	<li>Apply a thin but complete layer of quick-drying adhesive to one side of the 0.2&#160;mm acrylic and glue it to the bottom of the chamber. Hold the 0.2&#160;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.</li>
	<li>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.</li>
	<li>Using a 23 G, 1 in&#160;needle, poke a hole in the natural rubber on both sides of the chamber to facilitate insertion of the piezoelectric inkjet tip.</li>
</ol>
2. Experimental Preparation
Store stock lipid solution in chloroform in a -20 &#176;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&#160;of 25 mg/ml&#160;lipid solution is typically prepared in this protocol and will last for two months when stored at -20 &#176;C.
<ol>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Prepare a stock sucrose solution. In a 1.5-ml microcentrifuge tube, add 900 &#181;l of 300 mM sucrose and 100 &#181;l of 1% methylcellulose (MC). Methylcellulose is added to increase viscosity of the solution, which aids in jetting. Optional step: Add an optional 1 &#181;l 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.</li>
	<li>Draw the solution into a disposable 1&#160;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.</li>
	<li>Install a 0.22&#160;&#181;m 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&#160;mm diameter syringe filter unit was found to work best, but alternative filters as small as a 3&#160;mm diameter syringe filter can be used to reduce dead volume.</li>
	<li>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.</li>
</ol>
3. Readying the Equipment
<ol>
	<li>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.</li>
	<li>Determine the magnification and necessary lens combination to achieve the desired imaging. Here a 10X objective and 10X eyepiece are used.</li>
	<li>Use a high-speed camera (&#8805;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.</li>
	<li>Mount the infinity chamber onto the microscope stage. Secure the chamber by taping it into place on the stage.</li>
	<li>Carefully align the inkjet tip with the hole punctured in the natural rubber (see Figure&#160;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.</li>
	<li>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.</li>
	<li>Press gently on the plunger of the syringe assembly until a small droplet forms at the inkjet nozzle. This will provide some initial backpressure.</li>
	<li>Input the jetting parameters. For a trapezoidal bipolar wave, use the following parameters: 20 kHz pulse frequency, 3 &#181;sec rise time, 35 &#181;sec pulse duration, 3 &#181;sec 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.</li>
</ol>
4. Vesicle Generation
<ol>
	<li>Add 25-30 &#181;l lipid solution suspended in n-decane to the infinity chamber, covering the full surface of both wells.</li>
	<li>Add 25 &#181;l 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 &#181;l 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.</li>
	<li>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.</li>
	<li>When the inkjet is within ~200 &#181;m, 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.</li>
</ol>
5. Cleaning the Equipment
<ol>
	<li>Detach the syringe assembly from the microscope stage, and dispose of the 1&#160;ml plastic syringe and filter.</li>
	<li>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&#39;t fit securely on the aspiration pipette, cut a pipette tip to form an adapter.</li>
	<li>Dry the chamber with tissue. Place the infinity chamber in a 250&#160;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 &#176;C for 15 min.</li>
</ol>

<ol>
	<li>Liu, A. P., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+under+construction:+in+vitro+reconstitution+of+cellular+function.">Biology under construction: in vitro reconstitution of cellular function.</a> Nature reviews. Mol. Cell Biol. 10, 644-650 (2009).</li><li>Yeh, B. J., Lim, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology:+lessons+from+the+history+of+synthetic+organic+chemistry.">Synthetic biology: lessons from the history of synthetic organic chemistry.</a> Nat. Chem. Biol. 3, 521-525 (2007).</li><li>Richmond, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forming+giant+vesicles+with+controlled+membrane+composition,+asymmetry,+and+contents.">Forming giant vesicles with controlled membrane composition, asymmetry, and contents.</a> Proc. Natl. Acad. Soc. U.S.A. 108, 9431-9436 (2011).</li><li>Liu, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-induced+bundling+of+actin+filaments.">Membrane-induced bundling of actin filaments.</a> Nat. Phys. 4, 789-793 (2008).</li><li>Brown, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis+egg+extracts+provide+insight+into+scaling+of+the+mitotic+spindle.">Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle.</a> J. Cell Biol. 176, 765-770 (2007).</li><li>Loose, M., Fischer-Friedrich, E., Ries, J., Kruse, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulators+for+bacterial+cell+division+self-organize+into+surface+waves+in+vitro.">Spatial regulators for bacterial cell division self-organize into surface waves in vitro.</a> Science. 320, 789-792 (2008).</li><li>Angelova, M. I., Dimitrov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+electroformation.">Liposome electroformation.</a> Faraday Disc. Chem. Soc. 81, 301-311 (1986).</li><li>Bucher, P., Fischer, A., Luisi, L. P., Oberholzer, T., Walde, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+Vesicles+as+Biochemical+Compartments:+The+Use+of+Microinjection+Techniques.">Giant Vesicles as Biochemical Compartments: The Use of Microinjection Techniques.</a> Langmuir. 14, 2712-2721 (1998).</li><li>Shum, H. C., Lee, D., Yoon, I., Kodger, T., Weitz, D. 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C., Li, S., Malmstadt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Fabrication+of+Asymmetric+Giant+Lipid+Vesicles.">Microfluidic Fabrication of Asymmetric Giant Lipid Vesicles.</a> ACS Appl. Mater. Inter. 3, 1434-1440 (1021).</li><li>Hwang, W. L., Chen, M., Cronin, B., Holden, M. A., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+droplet+interface+bilayers.">Asymmetric droplet interface bilayers.</a> J. Am. Chem. Soc. 130, 5878-5879 (2008).</li><li>Stachowiak, J. C., Richmond, D. L., Li, T. H., Brochard-Wyart, F., Fletcher, D. 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A., Pott, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+unilamellar+vesicle+electroformation+from+lipid+mixtures+to+native+membranes+under+physiological+conditions.">Giant unilamellar vesicle electroformation from lipid mixtures to native membranes under physiological conditions.</a> Methods Enzymol. 465, 161-176 (2009).</li><li>Nishimura, K., Suzuki, H., Toyota, T., Yomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+control+of+giant+unilamellar+vesicles+prepared+from+inverted+emulsion+droplets.">Size control of giant unilamellar vesicles prepared from inverted emulsion droplets.</a> J. Colloid Interface Sci. 376, 119-125 (2012).</li><li>Teh, S. Y., Lin, R., Hung, L. H., Lee, A. 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No conflicts of interest declared.

Many techniques have been developed for vesicle generation, including electroformation, emulsion, and droplet generation14-16. 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 contents17,18, bringing vesicle models closer to biology. Furthermore, characterization and experimentation using microfluidic jetting has shown effective incorporation of oriented membrane proteins, membrane asymmetry, and encapsulation3,13.
This technique is straightforward, but includes some steps where caution should be taken. Both the acrylic sheet and natural rubber must form complete seals with the infinity chamber during fabrication. Otherwise, the infinity-shaped wells will leak and risk bilayer stability. During experimental preparation, the researcher must be sure to entirely evacuate the syringe assembly of air, initially and after attaching each component. Bubbles within this assembly create a compressible volume within the jet and mitigate or negate the jetting effect of the piezoelectric actuator. While readying the equipment, the main concern is damaging the fragile inkjet nozzle. Finally, the deposition of glucose requires the most attention during steps preceding vesicle generation, as this establishes the lipid bilayer for subsequent jetting.
Vesicle generation by microfluidic jetting is reliable and repeatable; however, discrepancies amongst inkjets require some familiarity and parameterization. In our experience, the introduction of the inkjet nozzle into the infinity chamber prior to jetting may displace up to several microliters of glucose depending on the nozzle dimensions, producing a slight bend in the bilayer away from the inkjet. By disproportionally dispersing the glucose solution when originally establishing the bilayer, this effect can be offset and a planar bilayer will result. This not only enhances bilayer stability but also allows for better control over vesicle formation. A 20 &#181;m orifice diameter of the inkjet was used to obtain results shown in this paper. An orifice diameter of 10-20 &#181;m is recommended. Minimization of vibrations is also recommended; simple rubber cutouts were used to support the microscope table and dampen laboratory vibrations.
Although this protocol is applicable to many lipids, DPhPC was chosen for its particular chemistry and high bilayer stability. Other primary lipids tested were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Comparatively, DPhPC had a stronger tendency to form a consistent droplet interface bilayer and to produce unilamellar vesicles. Upon initial formation, the bilayer in this protocol had an apparent thickness due to a nonplanar interface and the incorporation of oil. The shape of the aqueous droplets formed a curved bilayer, and this appeared as a thickness in a given field of view. DPhPC allowed the initial contact zone on the interface between the two droplets to expand, driving oil away from the bilayer. Due to this energetically favorable process, more and more of the contact zone between the two droplets became a bilayer, and the observable thickness decreased over time. This growth of the bilayer region was tested under variability of the length of the bilayer; the initial chamber design was adjusted slightly (the second generation design consisted of two circles of diameter 0.15 in&#160;separated by a center-to-center distance of 0.13 in) to necessitate smaller volumes and resulted in a smaller bilayer interface. Both the new and initial design allowed for accurate vesicle generation, yet neither design showed a dominant advantage in bilayer thinning. Another tested optimization was computer-controlled backpressure applied to the piezoelectric inkjet. While this gave a more quantitative control over the flow rate while jetting, it was not used throughout the majority of experimentation.
This method offers the combined advantages of several existing techniques. Multiple GUVs can be generated at high frequency (~200 Hz) due to the high concentration of lipid molecules, although rapid vesicle generation was not the focus of this work. Since this technique jets against a single lipid bilayer, membrane unilamellarity is expected and has been observed. Additionally, a wide range of solutions can be encapsulated independent of specific solute properties such as molecular weight or charge, thus enabling more potential applications17. Also, due to the size of vesicles formed (a range is possible of &#62;10 &#181;m to &#60;400 &#181;m), observation by conventional microscopy techniques is adequate13.
Microfluidic jetting can be applied to a variety of biological problems. One specific example is cellular biomechanics; the deformability of GUVs renders them an ideal tool to study the force generation and self assembly of encapsulated actin networks that showed interesting effects when assembled on the surface of a GUV4,19. Additional applications include drug delivery systems, cell-size bioreactors, and modular systems for synthetic biology, biophysics, and a variety of other fields in basic science, industry, and medicine where compartmentalized biomolecules are desired.

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&#39;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&#39;s plasma membrane. Formation of giant unilamellar vesicles (GUVs) by electroformation7, one of the most widely used techniques for GUV generation14,&#160;typically has a poor encapsulation yield due to its incompatibility with high salt buffer8. Electroformation also requires large sample volumes (&#62;100 &#956;l), 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:633px;" width="633">
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Name</td>
			<td style="width:163px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:160px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">Piezoelectric Inkjet</td>
			<td style="width:163px;">MicroFab Technologies</td>
			<td style="width:157px;">MJ-AL-01-xxx</td>
			<td style="width:160px;">xxx denotes orifice diameter in microns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Jet Drive III Controller</td>
			<td style="width:163px;">MicroFab Technologies</td>
			<td style="width:157px;">CT-M3-02</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">High-speed camera</td>
			<td style="width:163px;">Vision Research</td>
			<td style="width:157px;">MiroEX2</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">DPhPC lipid in chloroform</td>
			<td style="width:163px;">Avanti</td>
			<td style="width:157px;">850356C</td>
			<td style="width:160px;">Ordered in small aliquots in vials</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">33mm PVDF filters, 0.2 &#181;m</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">SLGV033RS</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">1ml syringes</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">14823434</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">n-Decane</td>
			<td style="width:163px;">Acros Organics</td>
			<td style="width:157px;">111871000</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Glucose</td>
			<td style="width:163px;">Acros Organics</td>
			<td style="width:157px;">410950010</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Sucrose</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:157px;">S7903-1KG</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Methylcellulose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">NC9084958</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:153px;">1/8&#34; Acrylic</td>
			<td style="width:163px;">McMaster Carr</td>
			<td style="width:157px;">8560K239</td>
			<td style="width:160px;">CAD designs for the infinity-shaped chamber are available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">0.2 mm Acrylic</td>
			<td style="width:163px;">Astra Products</td>
			<td style="width:157px;">Clarex clear 001</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Acrylic Cement</td>
			<td style="width:163px;">TAP Plastics</td>
			<td style="width:157px;">10693</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Loctite 495 Superglue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">NC9011323</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:153px;">Loctite 3494 UV Strengthening Adhesive</td>
			<td style="width:163px;">Strobels Supply</td>
			<td style="width:157px;">30765</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Natural rubber</td>
			<td style="width:163px;">McMaster Carr</td>
			<td style="width:157px;">85995K14</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">Custom stage</td>
			<td style="width:163px;">Home made</td>
			<td style="width:157px;">N/A</td>
			<td style="width:160px;">CAD designs are available upon request</td>
		</tr>
	</tbody>
</table>

lipid bilayer vesicle generation using microfluidic jetting

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 therapies1,2. 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.

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 the point of vesicle generation. The vortex travel increases with the voltage applied to the piezoelectric actuator, consistent with our expectations (Figure 2). Vesicle formation and representative jetted vesicles are shown in Figure 3. Figure 3a shows a representative image sequence for vesicle formation by microjetting. Following formation, vesicle stability tends to vary with vesicle diameter, where smaller vesicles were more stable.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51510/51510fig1highres.jpg" src="/files/ftp_upload/51510/51510fig1.jpg" /> 
Figure 1. Illustration of the technique and equipment. (a) Schematic of piezoelectric-driven jetting process against the droplet interface bilayer. Multiple pulses pushed out in rapid succession form a vortex ring structure that deforms the bilayer to produce GUVs. Bottom right depiction shows the location of glucose deposition and dimension of the well. Once lipid solution has been added to the infinity chamber, 25 &#181;l glucose is added to the outermost edges of the well, first at (1), then at (2). (b) The mounted syringe assembly, custom holding platform, and infinity chamber. The chamber is secured in place, and the tip of the inkjet is aligned with the hole in the natural rubber on the side of the well. (c) The complete microscope and custom stage assembly. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51510/51510fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51510/51510fig2highres.jpg" src="/files/ftp_upload/51510/51510fig2.jpg" /> 
Figure 2. Characterization of the Inkjet. (a) Rapid inkjet pulses at 20 kHz (50 pulses at 55 V pulse amplitude) overlap to form a single vortex ring. (b) Liquid jet front displacement as a function of time over pulse amplitude range (40-65 V) for fixed pulse number (200 pulses). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51510/51510fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51510/51510fig3highres.jpg" src="/files/ftp_upload/51510/51510fig3.jpg" /> 
Figure 3. Vesicle Generation. Images of the vesicle generation process and several vesicles produced. (a) Deformation of the droplet interface bilayer (DPhPC) produced by rapid pulses of solution cause the membrane to pinch off and form a GUV. (b) Many vesicles generated using microfluidic jetting.

Watch this Scientific Journal Video about Lipid Bilayer Vesicle Generation Using Microfluidic Jetting at JoVE.com

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

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.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

lipid-bilayer-vesicle-generation-using-microfluidic-jetting

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.8K Views.  University of Michigan. 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.

Video: Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...