This work was supported by a Research Award from the California Institute for Quantitative Biosciences (QB3), the Bridging the Gap Award from the Rogers Family Foundation, the UCSF/Sandler Foundation Program for Breakthrough Biomedical Research, a grant from BASF, and the NSF through the Faculty Early Career Development (CAREER) Program (DBI-1253293).

1. SU8 Master Fabrication
<ol>
	<li>Design the microfluidic structures for two layer fabrication using AutoCAD software and have the designs printed by a vendor on circuit board film with 10 &#181;m resolution. The details of device design are given in an attached reference11 and the channel geometries are shown in Figure 1. The layers should include alignment marks to help collocate features from each fabrication layer12.</li>
	<li>Place a pre-cleaned 3 inch diameter silicon wafer on a spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3035 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 2,000 rpm, providing a layer thickness of 50 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min. Allow the wafer to cool to RT before moving on to the next step.</li>
	<li>Expose the coated wafer to the 1st layer mask (Figure 2A) under a collimated 190 mW, 365 nm LED for 90 sec. After exposure, place the wafer on a 135 &#176;C hotplate for 1 min, then cool to RT before proceeding to the next step.</li>
	<li>Place the wafer on the spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-2050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 1,375 rpm, resulting in a layer that provides an additional thickness of 135 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min, then cool to RT before moving to the next step.</li>
	<li>Align the 2nd layer mask (Figure 2B) onto the geometry patterned in 1.3 and expose the coated wafer to a collimated 190 mW, 365 nm LED for 3 min. After exposure, place on a 135 &#176;C hotplate for 1 min, then cool to RT before proceeding to the next step.</li>
	<li>Develop the masks by immersing in a stirred bath of propylene glycol monomethyl ether acetate for 30 min. Wash the wafer in isopropanol and bake on a 135 &#176;C hotplate for 1 min. Place the developed master in a 100 mm Petri dish for PDMS molding.</li>
</ol>
2. PDMS Device Fabrication
<ol>
	<li>Prepare 10:1 PDMS by combining 50 g of silicone base with 5 g of curing agent in a plastic cup. Mix the contents with a rotary tool fitted with a stir stick. Degas the mixture inside a desiccator for 30 min, or until all air bubbles are removed.</li>
	<li>Pour the PDMS to give a thickness of 3 mm over the master and place back into the desiccator for further degassing. Once all bubbles are removed, bake the device at 60 &#176;C for 2 hr.</li>
	<li>Cut the device from the mold using a scalpel and place on a clean surface with the patterned side up. Cut the PDMS mold in half with a razor blade to separate Master 1 from Master 2 (Figure 3a). On the piece containing the 50 &#181;m fluid handling geometry imprinted by Master 1, punch the fluidic inlets and outlets with a 0.75 mm biopsy punch.</li>
	<li>Plasma treat the devices at 1 mbar O2 plasma for 60 sec in a 300 W plasma cleaner. Wet the surface of the unpunched piece of PDMS with a drop of DI water to temporarily retard PdMS-PDMS bonding and serve as a lubricant. While viewing through a stereo microscope, place Master 1 on Master 2 surface and slide the surfaces relatively until a mechanical lock is achieved when the recessed frames and protruding frames in Figure 3A mate.</li>
	<li>Place the device in a 60 &#176;C oven and bake the assembled device (Figure 3B) for two days at 60 &#176;C to evaporate the water and complete bonding.</li>
</ol>
3. Preparation of Reagents
<ol>
	<li>Fill 1 ml syringe with distilled water for the inner phase.</li>
	<li>Fill 1 ml syringe with HFE 7500 fluorinated oil with 1 wt. % biocompatible surfactant surfactant13 for the middle phase.</li>
	<li>Fill 10 ml syringe with 10 wt. % polyethylene glycol (PEG) in water solution containing 1 wt. % Tween 20 and 1 wt. % sodium dodecyl sulfate for the continuous phase.</li>
</ol>
4. System Preparation
<ol>
	<li>Place the microfluidic chip on the stage of an inverted microscope coupled with a digital camera capable of &#60; 100 &#181;sec shutter speeds.</li>
	<li>Mount all syringes on syringe pumps and attach 27 G needles. Attach ~ 30 cm lengths of PE-2 tubing on the needles and insert the loose ends into the appropriate punched holes in the device.</li>
	<li>Insert a 10 cm length of PE-2 into the exit port of the device and place the other end in a waste collection container.</li>
	<li>Prime the device by running the syringe pumps at high rates of speeds (2,000 &#181;l/min) until fluid in the tubing segments reaches the inlet ports of the device.</li>
</ol>
5. Emulsion Generation
<ol>
	<li>Focus the microscope on a region that contains the 50 &#181;m x 50 &#181;m orifice and the downstream exit channel.</li>
	<li>Set the syringe pumps to deliver fluid to the double emulsion generator at flow rates of 250 &#181;l/hr for the inner phase, 100 &#181;l/hr for the middle phase, and 700 &#181;l/hr for the continuous phase and wait 10 min for equilibration.</li>
	<li>Maintain the flow rates of the inner and middle phases at 250 &#181;l/hr and 100 &#181;l/hr, respectively. Set the flow rate of the outer phase at 1,050 &#181;l/hr. Wait 3-5 min for the double emulsions generation to stabilize under this set of flow conditions.</li>
	<li>Acquire 5 sec of video images at 30 Hz for offline processing via manual image analysis.</li>
	<li>Repeat 5.3 and 5.4 with the flow rates given in Table 1. The inner and middle phase flow rates are held constant and the carrier phase flow rate is varied by adjusting the setting of the syringe pump.</li>
</ol>

<ol>
	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, 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=Double-emulsion+drops+with+ultra-thin+shells+for+capsule+templates.">Double-emulsion drops with ultra-thin shells for capsule templates.</a> Lab Chip. 11 (18), 3162-3166 (2011).</li><li>Lim, S. W., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-throughput+sorting+of+microfluidic+drops+with+flow+cytometry.">Ultrahigh-throughput sorting of microfluidic drops with flow cytometry.</a> Lab Chip. 13 (23), 4563-4572 (2013).</li><li>Bernath, K., Hai, M., Mastrobattista, E., Griffiths, A. D., Magdassi, S., Tawfik, 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=In+vitro+compartmentalization+by+double+emulsions:+sorting+and+gene+enrichment+by+fluorescence+activated+cell+sorting.">In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.</a> Anal. Biochem. 325 (1), 151-157 (2004).</li><li>Seemann, R., Brinkmann, M., Pfohl, T., Herminghaus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+based+microfluidics.">Droplet based microfluidics.</a> Rep. Prog. Phys. 75 (1), 016601 (2012).</li><li>Bauer, W. A. C., Fischlechner, M., Abell, C., Huck, W. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+PDMS+microchannels+for+high-throughput+formation+of+oil-in-water+microdroplets+and+water-in-oil-in-water+double+emulsions.">Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions.</a> Lab Chip. 10 (14), 1814-1819 (2010).</li><li>Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., Weitz, 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=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li><li>Chang, F. C., Su, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+double+emulsification+utilizing+3D+PDMS+microchannels.">Controlled double emulsification utilizing 3D PDMS microchannels.</a> J. Micromech. Microeng. 18 (6), 065018 (2008).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, 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=High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li><li>Tran, T. M., Cater, S., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coaxial+flow+focusing+in+poly(dimethylsiloxane)+microfluidic+devices.">Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices.</a> Biomicrofluidics. 8 (1), 016502 (2014).</li><li>. Lithography Available from: <a target="_blank" href="https://www.memsnet.org/mems/processes/lithography.html">https://www.memsnet.org/mems/processes/lithography.html</a> (2015)</li><li>O'Donovan, B., Eastburn, D. J., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrode-free+picoinjection+of+microfluidic+drops.">Electrode-free picoinjection of microfluidic drops.</a> Lab Chip. 12 (20), 4029-4032 (2012).</li><li>Chang, F. C., Lin, H. H., Su, Y. 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The authors have nothing to disclose.

The double emulsion generating geometry described here is designed to mimic the physics of glass capillary devices8. In these, aligned cylindrical glass capillaries are used to create a three phase coaxial jet that is sheared into uniform double emulsion droplets. The function of our 3D PDMS device is dependent on the central alignment of small features formed with 50 &#181;m tall fabrication with carrier phase channels that are 320 &#181;m in total height. There is a significant potential for to misaligning t...

Double emulsions consist of droplets separated from a carrier phase by an intermediate, immiscible fluid layer, and are of particular interest due to their potential uses in industrial, pharmaceutical, and biological applications1. In some cases, the ability to encapsulate high value compounds in a double emulsion&#39;s core enables material to be protected and released in a controlled manner. For example, drugs may be encapsulated under solubility conditions not appropriate for the external carrier fluid2. Additionally, the intermediate oil layer can be used as a capsule template for the encapsulation and delivery of drugs, cosmetics, and nutrients3. In biology, double emulsions are also useful in high throughput screening because they allow a massive number of sub-nanoliter experiments to be carried out, then detected and sorted using a fluorescence-activated cell sorting (FACS) instrument4,5.
The design of double emulsions with desired performance characteristics requires the precise control of double emulsion size, composition, and uniformity. Although bulk emulsification processes, such as membrane emulsification, are used in industry, the resulting emulsions are highly polydisperse, exhibiting a wide variety of functional properties1. The field of droplet microfluidics is naturally suited the generation of monodisperse emulsions with carefully controlled composition6. Microfluidic double emulsion generation has been achieved with two main strategies, sequential drop making and glass capillary flow focusing. Double emulsions can be generated in planar PDMS devices using a two-step drop making process. First, aqueous-in-oil emulsions are created using a water-in-oil drop-making region of a device with hydrophobic channel walls. Next, the emulsion can be flowed or reinjected into a drop-making region with hydrophilic walls suited for oil in water drop-making4. However, hydrophilic surface treatment of PMDS requires an additional fabrication step and is often temporary7. The most controllable and repeatable method to form double emulsions is by co-axial flow focusing, a technique pioneered using glass capillary microfluidics, whereby a concentric jet containing the three phases is sheared through a small orifice to produce monodisperse droplets8. This technique allows for the production of droplets much smaller than the channel dimensions, with the precise size and composition of the double emulsion being a function of the flow rates of each phase. The large difference between droplet and channel size and the protective outer sheath flow prevents droplets from contacting the channel walls, rendering surface treatment unnecessary. However, such glass devices require custom fabrication of tapered capillary tips, along with careful assembly and sealing. Previous investigators have used 3D soft lithography to generate double emulsions using flow focusing physics, but these devices produced emulsions with diameters &#62; 150 &#181;m 9,10, roughly an order of magnitude larger than objects typically sorted with FACS. An attractive alternative would include the robust functionality and small droplet generation of glass capillary coaxial flow focusing with the ease of manufacture of PDMS soft lithography.
In this paper, we describe a double emulsion generator that uses co-axial flow focusing to produce &#8804; 50 &#181;m emulsions and is constructed entirely using 3D soft lithography11. Our device uses a clamshell approach to fabricate devices that includes a small shearing channel (Figure 1) to approximate the emulsion formation processes in a pulled glass capillary nozzle. More importantly, these devices require no specific surface treatment, and the all polymer construction provides easy and repeatable fabrication scalable to a large number of duplicate devices. Here, we outline the design, fabrication, and testing of the double emulsion generator. Double emulsion generation is shown to be robust and repeatable down to droplet diameters of 14 &#181;m. The coupling of functionality with ease of fabrication makes this device an appealing option for development of new double emulsion applications.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
	</tbody>
</table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

The double emulsion generator consists of a co-axial flow focusing device created using 3D PDMS fabrication (Figure 1A). The geometry enables that formation of a three-phase co-axial jet to be sheared into a square, 50 &#181;m x 50 &#181;m orifice, allowing the formation of water / oil / water double emulsions (Figure 1B, Figure 1C). The inner aqueous phase and the middle oil phase are brought together at a junction with channel dimension...

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

Research

JoVE Journal

Bioengineering

16.0K Views.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

Video: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

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.

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 ...

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 ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

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 ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...