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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Centrifuge tube</td>
			<td>Solarbio, Beijing, China</td>
			<td></td>
			<td>5 mL &#38; 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 &#956;m, OD: 460 &#956;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&#39;s 
			Mod. Of Eagle&#39;s Medium/Ham&#39;s F12 
			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 &#215; 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-co-glycolic acid)</td>
			<td>Sigma-Aldrich Co. Ltd, St. Louis, USA</td>
			<td>lot# MKCF9651</td>
			<td>66&#8211;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 &#38; 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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

2.7K 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

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Reconfigurable Microfluidic Channel with Pin-discretized Sidewalls

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or reactions. A microfluidic channel that deforms even after fabrication while retaining the channel shape enables high spatiotemporal reconfigurability. This reconfigurability is required in such key microfluidic functions that are difficult to achieve in existing "reconfigurable" or "integrated" microfluidic systems. We describe a method for the fabrication of a microfluidic channel with a deformable sidewall consisting of a laterally aligned array of the ends of rectangular pins. Actuating the pins in their longitudinal directions changes the pins' end positions, and thus, the shape of discretized channel sidewalls.Pin gaps can cause unwanted leakage or adhesion to adjacent pins caused by meniscus forces. To close the pin gaps, we have introduced hydrocarbon-fluoropolymer suspension-based gap filler accompanied by an elastomeric barrier. This reconfigurable microfluidic device can generate strong temporal in-channel displacement flow, or can stop the flow in any region of the channel. This feature will facilitate, on demand, the handling of cells, viscous liquids, gas bubbles, and non-fluids, even if their existence or behavior is unknown at the time of fabrication.

A microfluidic channel with deformable sidewalls offers flow control, particle handling, channel dimension customization and other reconfigurations while in use. We describe a method for fabricating a microfluidic channel with sidewalls made of an array of pins that allows their shape to change.

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...