Name: fabricating highly open porous microspheres (hopms) via microfluidic technology
Uploaded: 2022-05-16T15:00:00
Description: Watch this Scientific Journal Video about Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology at JoVE.com

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 te...

<ol>
	<li>Kankala, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+porous+microcarriers+for+minimally+invasive+in+situ+skeletal+muscle+cell+delivery.">Highly porous microcarriers for minimally invasive in situ skeletal muscle cell delivery.</a> Small. 15 (25), 1901397 (2019).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+endothelialized+hepatic+tumor+microtissues+for+drug+screening.">Modeling endothelialized hepatic tumor microtissues for drug screening.</a> Advanced Science. 7 (21), 2002002 (2020).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tripeptide-based+macroporous+hydrogel+improves+the+osteogenic+microenvironment+of+stem+cells.">Tripeptide-based macroporous hydrogel improves the osteogenic microenvironment of stem cells.</a> Journal of Materials Chemistry B. 9 (30), 6056-6067 (2021).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA+hybrid+porous+microspheres+as+human+periodontal+ligament+stem+cell+delivery+carriers+for+periodontal+regeneration.">PLGA hybrid porous microspheres as human periodontal ligament stem cell delivery carriers for periodontal regeneration.</a> Chemical Engineering Journal. 420, 129703 (2021).</li><li>Wei, P., Xu, Y., Zhang, H., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continued+sustained+insulin-releasing+PLGA+nanoparticles+modified+3D-printed+PCL+composite+scaffolds+for+osteochondral+repair.">Continued sustained insulin-releasing PLGA nanoparticles modified 3D-printed PCL composite scaffolds for osteochondral repair.</a> Chemical Engineering Journal. 422, 130051 (2021).</li><li>Sang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+chitosan/polyethylene+glycol/multi-walled+carbon+nanotube+composite+scaffolds+for+neural+tissue+engineering.">Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering.</a> Journal of Zhejiang University-Science B. 23 (1), 58-73 (2022).</li><li>Ghasemian, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+characterization+within+a+spinner+flask+and+a+rotary+wall+vessel+for+stem+cell+culture.">Hydrodynamic characterization within a spinner flask and a rotary wall vessel for stem cell culture.</a> Biochemical Engineering Journal. 157, 107533 (2020).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+co-injection+of+modular+micro-muscular+and+micro-vascular+tissues+improves+in+situ+skeletal+muscle+regeneration.">Minimally invasive co-injection of modular micro-muscular and micro-vascular tissues improves in situ skeletal muscle regeneration.</a> Biomaterials. 277, 121072 (2021).</li><li>Kang, S. 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I., Bancroft, G. N., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+three-dimensional+cell/polymer+constructs+for+bone+tissue+engineering+in+a+spinner+flask+and+a+rotating+wall+vessel+bioreactor.">Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor.</a> Journal of Biomedical Materials Research. 62 (1), 136-148 (2002).</li><li>Kim, T. K., Yoon, J. J., Lee, D. S., Park, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+foamed+open+porous+biodegradable+polymeric+microspheres.">Gas foamed open porous biodegradable polymeric microspheres.</a> Biomaterials. 27 (2), 152-159 (2006).</li><li>Wang, C. Y., Liao, H. F., Sheu, D. 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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 ...

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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 ...

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 ...

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 ...

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 ...

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.), ...

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 ...

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 ...

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 ...

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 ...