SCL, YW, RKK, and AZC acknowledge financial support from the National Natural Science Foundation of China (NSFC, 32071323, 81971734, and U1605225) and Program for Innovative Research Team in Science and Technology in Fujian Province University. YSZ was neither supported by any of these programs nor received payment of any type; instead, support from the Brigham Research Institute is acknowledged.

1. Preparation of solutions
<ol>
	<li>Prepare the PVA stock solution in advance by heating the PVA solution in a 80 &#176;C water bath and subsequently placing it in the refrigerator at 4 &#176;C. Cool to room temperature (RT) for experimental usage.</li>
	<li>Prepare the emulsion-precursor by adding the aqueous gelatin solution (1 mL, 7.5%, w/v) to the organic phase of PLGA (2 mL, 2%, w/v in dichloromethane, DCM) (see Table of Materials). 
	&#8203;NOTE: Generally, microfluidic technology involves different phases to form the microspheres. The continuous or collecting phase comprises an aqueous poly(vinyl alcohol) (PVA, 600 mL, 1%, w/v).</li>
</ol>
2. Droplet microfluidic device assembly and fabrication of PLGA-HOPMs
NOTE: The consumable items required for this assembly are listed in the Table of Materials.
<ol>
	<li>Customize the droplet generator assembly.
	<ol>
		<li>Construct a co-flow microfluidic generator using a glass capillary (OD, 1 mm), PVC tubes (ID, 1 mm), and a 26 G dispensing needle.</li>
		<li>Connect the glass capillary to the end of the PVC tube and then pierce with a needle at the connection of the above structure.</li>
		<li>Place the other end of the PVC tube in the continuous phase during the droplet generation. Using UV glue, seal the gaps at the joint after curing. 
		NOTE: The needle needs to be bent curved about 45&#176; for convenient piercing at the PVC tube, and the needlepoint stretched into the capillary tube to form the coaxial structure.</li>
	</ol>
	</li>
	<li>Prepare the droplet microfluidic platform.
	<ol>
		<li>Use two syringe pumps, as shown in Figure 1. Use a 50 mL syringe to load the continuous phase and a 5 mL syringe for emulsion formation.</li>
		<li>Set the flow rates of the continuous and dispersion phases at 2 mL/min and 0.08 mL/min, respectively.</li>
		<li>Place a 500 mL beaker in an ice bath and fill it with a pre-cooled PVA aqueous solution (step 1.1). 
		NOTE: The end of the glass capillary must be immersed in the collecting phase. It is suggested to stir (1 h, 60 rpm) the supernatant of the collecting phase with the glass rod after the emulsion-droplets formation in the platform. Emulsion droplets are produced at the tip of the needlepoint. In general, the gauge of the needle influences the size of PMs, in which the lesser gauge corresponds to the smaller size of PMs. Moreover, the pore diameter is mainly correlated to the porogen concentration, capable of increasing with appropriate gelatin concentration1.</li>
	</ol>
	</li>
	<li>Perform the emulsification and droplet generation.
	<ol>
		<li>Prepare the emulsion following the steps below.
		<ol>
			<li>Prepare the emulsion by immediately decanting the aqueous gelatin solution into the DCM solution of PLGA (step 1.2) and emulsifying using the ultrasonic device (see Table of Materials).</li>
			<li>Adjust the ultrasonic power to 400 W, and set the total processing time as 90 s (interval 2 s, ultrasonic treatment 1 s), accompanied by constantly changing the probe&#39;s position manually.</li>
			<li>Stabilize the resultant emulsion for syringe suction and droplet generation in approximately 20 min. 
			NOTE: Place the probe of the ultrasonic cell breaker in the oil-water interface. It is suggested not to let the ultrasonic probe contact the container.</li>
		</ol>
		</li>
		<li>Perform the droplet generation.
		<ol>
			<li>Load the prepared emulsion (step 2.3.1) rapidly in the 5 mL syringe of the microfluidic platform after ultrasonic treatment.</li>
			<li>Introduce the unstable W/O emulsion (which is in the course of phase-separation) into the microfluidic device as the discontinuous phase. At the same time, use the aqueous solution of PVA as the continuous phase.</li>
			<li>Collect the microspheres containing the emulsion at the bottom of the collecting beaker (PVA, 1%, w/v) after injecting proper portions of the emulsion into the microfluidic device.</li>
			<li>Place the sample at 4 &#176;C overnight for further stabilization.</li>
		</ol>
		</li>
		<li>Rinse and lyophilize the prepared microspheres (PLGA-HOPMs) following the steps below. 
		&#8203;NOTE: The generated PLGA-HOPMs contain arbitrary pores on the interior and surface.
		<ol>
			<li>Store the microspheres at 4 &#176;C before normalizing to RT and stir by a glass rod (1 h, 60 rpm) to eliminate the residual DCM.</li>
			<li>Filtrate the collecting phase in the 500 mL beaker carefully, and wash the collected microspheres thrice with deionized water to wash off the residual PVA on the surface of PMs.</li>
			<li>Place the PLGA microspheres in the 37 &#176;C water bath for 30 min to dissolve the gelatin wrapped in the PLGA backbone.</li>
			<li>Rinse the resultant PLGA-HOPMs twice with the deionized water to remove the residual gelatin and pre-cool for lyophilization at -80 &#176;C for 24 h.</li>
			<li>Store the dry PLGA-HOPMs at -20 &#176;C for further experiments.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Scanning electron microscope (SEM) imaging
<ol>
	<li>Deposit PLGA-HOPMs on the sample stage of SEM (see Table of Materials) at RT and put the sample stage into the sputter cell of the magnetron sputter. Switch on the transformer and magnetron one by one. Introduce the sample to the SEM after being sputter-coated with gold for 1 min.</li>
	<li>Obtain the SEM images by setting the acceleration voltage at 5 kV.</li>
	<li>Additionally, quantify the particle size of PLGA-HOPMs using 100 PMs at the variable field of SEM imaging. Measure the representative result to quantify the pore size distribution.
	<ol>
		<li>Open the graphics with Image J and use the &#34;straight line&#34; to match the scale bar of the SEM image, which makes the software identify pixel to length.</li>
		<li>Click on analyze &#62; set scale, set the &#34;known distance&#34; to the scale bar of the SEM image, and click on global &#62; OK.</li>
		<li>Click on analyze &#62; Tools &#62; ROI manager... to measure the pore or particle size of PLGA PMs and click on add it. Measure the number of samples based on the requirement.</li>
		<li>Click on Measure to obtain the quantitative data of the graphics for further calculation.</li>
	</ol>
	</li>
</ol>
4. Cell culture and fluorescence imaging
NOTE: Rat bone marrow mesenchymal stem cells (BMSCs) were used for the present work. The cells were obtained from a commercial source (see Table of Materials).
<ol>
	<li>Culture the cells in Dulbecco&#39;s modified Eagle medium supplemented with L-glutamine (DMEM/F-12), 10% (v/v) fetal bovine serum, and 1% penicillin/streptomycin. Incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>Passage the rat BMSCs in a 1:2 ratio after reaching 90% of confluence.</li>
	<li>Perform cell adhesion following the steps below.
	<ol>
		<li>Incubate the PLGA-HOPMs with ethyl alcohol (5 mL, 70%, v/v) and centrifuge at 500 x g for 10 min at RT to sterilize.</li>
		<li>Wash the sterilized PLGA-HOPMs twice with phosphate buffer solution.</li>
		<li>Incubate the HOPMs (five in number) with the cell suspension (5 mL, 1 x 105 cells/mL) in a sterile centrifuge tube (50 mL) under dynamic culture for BMSCs adhesion to PLGA-HOPMs.</li>
		<li>Perform the dynamic culture in a thermostatic aseptic shaker (37 &#176;C, 90 rpm) by placing a sealed 50 mL centrifuge tube in the shaker (see Table of Materials). 
		NOTE: The dynamic culture intends to enhance the adherence rate of BMSCs on the scaffolds, rather than directly incubating the PMs and cells onto the plastic plate. The cells and PLGA PMs are added into a 50 mL sterile centrifuge tube, incubating the tube at a thermostatic aseptic shaker (37 &#176;C, 90 rpm) and changing the media every 2 days.</li>
	</ol>
	</li>
	<li>Perform the live and dead dual-stain assay.
	<ol>
		<li>Rinse the cell-laden PMs thrice for eluting BMSCs without attachment to PLGA-HOPMs.</li>
		<li>Place the cell-laden PMs on the 35 mm glass-bottom plate. Add 1 mL of staining buffer (see Table of Materials), including 1 &#181;L of calcein-AM and propidium iodide (PI), respectively, to the PBS-prewashed sample.</li>
		<li>Assess the samples by confocal laser scanning microscopy (see Table of Materials). Switch on the excitation light of 488 nm and 552 nm at the corresponding detector. For z-overlay analysis, set the z-wide to capture the different layers of the sample by z-axis movement of the microscope. 
		NOTE: It is worth noting that the other stains can be appropriately employed for analysis.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article describes an efficient strategy to fabricate PLGA-based architectures, namely the PLGA-HOPMs. It is to be noted that several critical steps must be taken carefully, including avoiding solvent volatilization of PLGA and gentle adjustment of the ultrasonic power to the target position during the preparation of the emulsion. In addition, the liquid outlet of the 20 mL syringe can be adjusted to a certain extent to solve the phase separation of emulsified precursors. However, a limitation is that, since the stat...

Cell-laden microspheres offer favorable advantages, such as enhanced cell retention capacity in situ, efficient delivery of cells, and subsequent ability of cell proliferation in vivo1. To date, numerous investigations have been put forward for developing a successful scaffolding structure to support a conducive environment for cells for tissue regeneration or drug screening applications2. However, the hypoxia environment is oftentimes inevitable in the interiors due to insufficient supplies of nutrients/oxygen and metabolic waste accumulation3. To overcome these problems, highly porous microspheres (PMs) have been developed using various biomaterials4,5,6. Additionally, in dynamic culture, the scaffolds suffer from excessive shear stress7, and the unstable state of the culture medium might break the fidelity of PMs. Alternatively, poly(lactic-co-glycolic acid) (PLGA) could be used to process PMs with good mechanical strength for dynamic culture1. For example, we demonstrated co-injection of mouse myoblast (C2C12)-laden PLGA highly open PMs (HOPMs) and human umbilical vein endothelial cell (HUVEC)-laden poly(ethylene glycol) hollow microrods to heal volumetric muscle loss, achieving remarkable improvement of in situ skeletal muscle regeneration8.
Notably, PMs are characterized by large surface areas and high porosities, which is of specific interest for cell adhesion and growth towards minimally invasive cell delivery9. In view of these aspects, various biocompatible materials have been employed to fabricate the PMs10,11. These designable PMs cocultured with cells offer excellent adhesion, considerable mechanical strength, and highly interconnected windows, which could improve cell proliferation for repairing damaged tissues12. In this regard, various technologies have also been developed to fabricate porous spheres13,14. On the one hand, PMs were produced using gas-forming agents, such as NH4HCO3, which were restrained due to insufficient interconnectivity15,16,17. On the other hand, PMs were directly sheared after emulsification, which led to polydisperse PMs18. In the end, the droplet microfluidic technology based on the emulsion-templating approach is perhaps an efficient method for constructing PMs, as it often results in uniform-sized particles19. Notably, the morphological attributes of the microspheres often depend on the quality of the generated emulsion droplets (i.e., water-in-oil, W/O, or oil-in-water, O/W), which may significantly affect the attributes of the biomaterials20. It is worth noting that the predesigned microfluidic platform can be applied to generate the microfibers or microspheres. In an instance, Yu et al. demonstrated the production of cell-laden microfibrous structures based on capillary-based microfluidic platforms, which could be used to assemble cellular networks for mimicking natural tissues21. In another instance, Ye et al. fabricated photonic crystal microcapsules by the template replication of silica colloidal crystal beads through microfluidic technologies, which could overcome many limitations of current techniques that require complex labeling and specific apparatus22.
Indeed, the rationale behind the utilization of this technique is due to various advantages, such as being facile in nature, requiring no sophisticated equipment, and its convenience in synthesizing uniform-sized PMs for cell delivery and regenerative medicine applications. In this context, with predesigned components of emulsion-templating, PMs with high porosities and interconnectivity can be conveniently obtained from a microfluidic device assembled from poly(vinyl chloride) (PVC) tubing, a glass capillary, and a needle. A W/O emulsion-precursor is prepared by homogenizing an aqueous solution of gelatin and an organic solution of PLGA. By selectively injecting the applicable portion of the emulsion into the microfluidic platform, the PMs with uniform particle sizes and interconnected pores throughout the surface to the interior are fabricated. The present protocol aims to fabricate the PLGA-HOPMs by emulsion-templating in the microfluidic platform. It is believed that this protocol allows reproducible production of PLGA-HOPMs and will potentially be applicable in their related fields of tissue engineering and drug screening.

<table><tbody><tr><td>Centrifuge tube</td><td>Solarbio, Beijing, China</td><td></td><td>5 mL &amp;amp; 50 mL (sterility)</td></tr><tr><td>Confocal laser scanning microscopy</td><td>Leica, Wetzlar, Germany</td><td></td><td>TCS SP8</td></tr><tr><td>Dichloromethane</td><td>Sinopharm Chemical Reagent Co., Ltd, Shanghai, China</td><td>20161110</td><td>Research Grade</td></tr><tr><td>Dispensing needle</td><td>Kindly, Shanghai, China</td><td></td><td>26 G, ID: 250 &amp;mu;m, OD: 460 &amp;mu;m</td></tr><tr><td>DMEM/F-12</td><td>Gibco; Life Technologies Corporation, Calsbad, USA</td><td>15400054</td><td>DMEM/F-12 50/50, 1x (Dulbecco&#039;s&lt;br/&gt; Mod. Of Eagle&#039;s Medium/Ham&#039;s F12&lt;br/&gt; 50/50 Mix) with L-glutamine</td></tr><tr><td>Ethyl alcohol</td><td>Sinopharm Chemical Reagent Co., Ltd, Shanghai, China</td><td>20210918</td><td>Research Grade</td></tr><tr><td>Ethyl-enediaminetetraacetic acid (EDTA)-trypsin</td><td>Biological Industries, Kibbutz Beit-Haemek, Isra</td><td></td><td>Trypsin (0.25%), EDTA (0.02%)</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biological Industries, Kibbutz Beit-Haemek, Isra</td><td></td><td>Research Grade</td></tr><tr><td>Freeze drier</td><td>Bilon, Shanghai, China</td><td></td><td>FD-1B-50</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich Co. Ltd, St. Louis, USA</td><td>lot# SZBF2870V</td><td>From porcine skin, Type A</td></tr><tr><td>Glass bottom plate</td><td>Biosharp, Hefei, China</td><td></td><td>BS-15-GJM, 35 mm</td></tr><tr><td>Glass capillary</td><td>Huaou, Jiangsu, China</td><td></td><td>0.9-1.1 &amp;times; 120 mm</td></tr><tr><td>Incubator shaker</td><td>Zhicheng, Shanghai, China</td><td>ZWYR-200D</td><td></td></tr><tr><td>Live dead kit cell imaging kit</td><td>Solarbio, Beijing, China</td><td>60421211112</td><td>Green fluorescence in live cells (ex/em 488 nm/515 nm). Red fluorescence in dead cells (ex/em 570 nm/602 nm)</td></tr><tr><td>Low-speed centrifuge</td><td>Xiangyi, Hunan, China</td><td></td><td>TD5A</td></tr><tr><td>Magnetron sputter</td><td>Riye electric Co. Ltd, Suzhou, China</td><td>MSP-2S</td><td></td></tr><tr><td>Microflow injection pump</td><td>Harvard Apparatus, Holliston, USA</td><td></td><td>Harvard Pump 11 Plus</td></tr><tr><td>Penicillin-streptomycin</td><td>Biological Industries, Kibbutz Beit-Haemek, Isra</td><td>2135250</td><td>Research Grade</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Servicebio Technology Co.,Ltd. Wuhan, China</td><td>GP21090181556</td><td>PBS 1x, culture grade, no Calcium, no Magnesium</td></tr><tr><td>Poly(lactic-&lt;em&gt;co&lt;/em&gt;-glycolic acid)</td><td>Sigma-Aldrich Co. Ltd, St. Louis, USA</td><td>lot# MKCF9651</td><td>66&amp;ndash;107 kDa, lactide:glycolide 75:25</td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma-Aldrich Co. Ltd, St. Louis, USA</td><td>lot# MKCK4266</td><td>13-13 kDa, 98% Hydrolyzed</td></tr><tr><td>PVC tube</td><td>Shenchen, Shanghai, China</td><td></td><td>Inner diameter, ID: 1 mm</td></tr><tr><td>Rat bone marrow mesenchyml stem cells</td><td>Procell, Wuhan, China</td><td></td><td></td></tr><tr><td>Scanning electron microscope</td><td>Phenom pure, Eindhoven, Netherlands</td><td></td><td>Set acceleration voltage at 5 kV</td></tr><tr><td>Syrine for medical purpose</td><td>Kindly, Shanghai, China</td><td></td><td>5 mL &amp;amp; 50 mL (with the needle)</td></tr><tr><td>Temperature water bath</td><td>Mingxiang, Shenzhen, China</td><td></td><td>36 W</td></tr><tr><td>Transformer</td><td>Riye electric Co. Ltd, Suzhou, China</td><td>SZ-2KVA</td><td></td></tr><tr><td>Ultrasonic cell breaker</td><td>JY 92-IID, Scientz, Ningbo, China</td><td></td><td>JY 92-IID</td></tr><tr><td>UV curing glue</td><td>Zhuolide, Foshan, China</td><td></td><td>D-3100</td></tr></tbody></table>

fabricating highly open porous microspheres (hopms) via microfluidic technology

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned tissues due to convenience in the packaging of cells, improved cell retention, and minimal invasiveness. Moreover, the porous conformation of these microscale carriers could enhance the medium exchange and improve the level of nutrients and oxygen supplies. The present study illustrates the convenient fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (PLGA-HOPMs) by the facile microfluidic technology for cell delivery applications. The resultant monodispersed PLGA-HOPMs possessed particle sizes of ~400 &#181;m and open pores of ~50 &#181;m with interconnecting windows. Briefly, the emulsified oil droplets (PLGA solution in dichloromethane, DCM), wrapped with the 7.5% (w/v) gelatin aqueous phase, were introduced into the 1% (w/v) continuous flowing poly(vinyl alcohol) (PVA) aqueous solution through the coaxial nozzle in the customized microfluidic setup. Subsequently, the microspheres were subjected to solvent extraction and lyophilization procedures, resulting in the production of HOPMs. Notably, various formulations (concentrations of PLGA and porogen) and processing parameters (emulsifying power, needle gauge, and flow rate of dispersed phase) play crucial roles in the qualities and characteristics of the resulting PLGA HOPMs. Moreover, these architectures might potentially encapsulate various other biochemical cues, such as growth factors, for extended drug discovery and tissue regeneration applications.

Based on previous work that optimized the major parameters1, PLGA was dissolved in the evaporable DCM solvent. The primary W/O emulsion was prepared by homogenizing with gelatin under ultrasonic probe treatment. The customized co-flow fluidic structure was simplistically assembled, in which a syringe was employed to introduce the flows constantly. Furthermore, sufficient rinsing procedures were carried out to eliminate PVA and gelatin of PLGA microspheres (Figure 1A, ...

Watch this Scientific Journal Video about Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology at JoVE.com

Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned ...

fabricating-highly-open-porous-microspheres-hopms-via-microfluidic

Research

JoVE Journal

Bioengineering

2.8K Views.  Huaqiao University. The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

Video: Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Chemistry

Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are resistant to low molecular-weight oil components, unlike traditional polydimethylsiloxane (PDMS) devices. Here, we demonstrate a facile method for making a device with this property, and we use the product of this protocol for examining the pore-scale mechanisms by which foam recovers crude oil. A pattern is first designed using computer-aided design (CAD) software and printed on a transparency with a high-resolution printer. This pattern is then transferred to a photoresist via a lithography procedure. PDMS is cast on the pattern, cured in an oven, and removed to obtain a mold. A thiol-ene crosslinking polymer, commonly used as an optical adhesive (OA), is then poured onto the mold and cured under UV light. The PDMS mold is peeled away from the optical adhesive cast. A glass substrate is then prepared, and the two halves of the device are bonded together. Optical adhesive-based devices are more robust than traditional PDMS microfluidic devices. The epoxy structure is resistant to swelling by many organic solvents, which opens new possibilities for experiments involving light organic liquids. Additionally, the surface wettability behavior of these devices is more stable than that of PDMS. The construction of optical adhesive microfluidic devices is simple, yet requires incrementally more effort than the making of PDMS-based devices. Also, though optical adhesive devices are stable in organic liquids, they may exhibit reduced bond-strength after a long time. Optical adhesive microfluidic devices can be made in geometries that act as 2-D micromodels for porous media. These devices are applied in the study of oil displacement to improve our understanding of the pore-scale mechanisms involved in enhanced oil recovery and aquifer remediation.

The goal of this procedure is to easily and rapidly produce a microfluidic device with customizable geometry and resistance to swelling by organic fluids for oil recovery studies. A polydimethylsiloxane mold is first generated, and then used to cast the epoxy-based device. A representative displacement study is reported.

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are ...

Environment

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. Customized channel layout designs are necessary for specific functions and integrated performance of microfluidic devices in numerous biomedical and chemical applications (e.g., cell culture, biosensing, chemical synthesis, and liquid handling). Owing to the nature of molding approaches using silicon wafers with photoresist layers patterned by photolithography as master molds, the microfluidic channels commonly have regular cross sections of rectangular shapes with identical heights. Typically, channels with multiple heights or different geometric sections are designed to possess particular functions and to perform in various microfluidic applications (e.g., hydrophoresis is used for sorting particles and in continuous flows for separating blood cells6,7,8,9). Therefore, a great deal of effort has been made in constructing channels with various sections through multiple-step approaches like photolithography using several photoresist layers and assembly of different PDMS thin sheets. Nevertheless, such multiple-step approaches usually involve tedious procedures and extensive instrumentation. Furthermore, the fabricated devices may not perform consistently and the resulted experimental data may be unpredictable. Here, a one-step approach is developed for the straightforward fabrication of microfluidic channels with different geometric cross sections through PDMS sequential wet etching processes, that introduces etchant into channels of planned single-layer layouts embedded in PDMS materials. Compared to the existing methods for manufacturing PDMS microfluidic channels with different geometries, the developed one-step approach can significantly simplify the process to fabricate channels with non-rectangular sections or various heights. Consequently, the technique is a way of constructing complex microfluidic channels, which provides a fabrication solution for the advancement of innovative microfluidic systems.

Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching.

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. ...

Engineering

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Biology

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e.,&#160;annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications.

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that ...