Name: microfluidic synthesis of microgel building blocks for microporous annealed particle scaffold
Uploaded: 2022-06-16T14:40:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold at JoVE.com

The authors would like to acknowledge Joe de Rutte and the Di Carlo Lab at the University of California, Los Angeles, for their assistance with the original microfluidic device design that the reported device was developed from, as well as their early guidance in PDMS device fabrication and troubleshooting. Figure schematics were created with Biorender.com.

<ol>
	<li>Griffin, D. R., Weaver, W. M., Scumpia, P. O., di Carlo, D., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+wound+healing+by+injectable+microporous+gel+scaffolds+assembled+from+annealed+building+blocks.">Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks.</a> Nature Materials. 14 (7), 737-744 (2015).</li><li>Koh, J., 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=Enhanced+in+vivo+delivery+of+stem+cells+using+microporous+annealed+particle+scaffolds.">Enhanced in vivo delivery of stem cells using microporous annealed particle scaffolds.</a> Small. 15 (39), 1903147 (2019).</li><li>Pruett, L. J., Jenkins, C. H., Singh, N. S., Catallo, K. J., Griffin, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+microislands+in+microporous+annealed+particle+scaffolds+for+accelerated+diabetic+wound+healing.">Heparin microislands in microporous annealed particle scaffolds for accelerated diabetic wound healing.</a> Advanced Functional Materials. 31 (35), 2104337 (2021).</li><li>Darling, N. J., Sideris, E., Hamada, N., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+and+spatially+patterned+microporous+annealed+particle+(MAP)+hydrogels+for+tissue+repair+applications.">Injectable and spatially patterned microporous annealed particle (MAP) hydrogels for tissue repair applications.</a> Advanced Science. 5 (11), 1801046 (2018).</li><li>Isaac, A., 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=Microporous+bio-orthogonally+annealed+particle+hydrogels+for+tissue+engineering+and+regenerative+medicine.">Microporous bio-orthogonally annealed particle hydrogels for tissue engineering and regenerative medicine.</a> ACS Biomaterials Science and Engineering. 5 (12), 6395-6404 (2019).</li><li>Pruett, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+microporous+annealed+particle+hydrogel+for+long-term+vocal+fold+augmentation.">Development of a microporous annealed particle hydrogel for long-term vocal fold augmentation.</a> Laryngoscope. 130 (10), 2432-2441 (2020).</li><li>Nih, L. R., Sideris, E., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+microporous+annealing+particle+(MAP)+hydrogels+in+the+stroke+cavity+reduces+gliosis+and+inflammation+and+promotes+NPC+migration+to+the+lesion.">Injection of microporous annealing particle (MAP) hydrogels in the stroke cavity reduces gliosis and inflammation and promotes NPC migration to the lesion.</a> Advanced Materials. 29 (32), (2017).</li><li>Griffin, D. R., 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=Activating+an+adaptive+immune+response+from+a+hydrogel+scaffold+imparts+regenerative+wound+healing.">Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing.</a> Nature Materials. 20 (4), 560-569 (2021).</li><li>Sideris, E., 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=Particle+hydrogels+based+on+hyaluronic+acid+building+blocks.">Particle hydrogels based on hyaluronic acid building blocks.</a> ACS Biomaterials Science and Engineering. 2 (11), 2034-2041 (2016).</li><li>Schaeffer, C., 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=Injectable+microannealed+porous+scaffold+for+articular+cartilage+regeneration.">Injectable microannealed porous scaffold for articular cartilage regeneration.</a> Annals of Plastic Surgery. 84, 446-450 (2020).</li><li>Pruett, L., Ellis, R., McDermott, M., Roosa, C., Griffin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+heterogeneous+epidermal+growth+factor+release+from+microporous+annealed+particle+(MAP)+hydrogel+for+improved+wound+closure.">Spatially heterogeneous epidermal growth factor release from microporous annealed particle (MAP) hydrogel for improved wound closure.</a> Journal of Materials Chemistry B. 9 (35), 7132-7139 (2021).</li><li>Jivan, F., Alge, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-orthogonal,+site-selective+conjugation+of+recombinant+proteins+to+microporous+annealed+particle+hydrogels+for+tissue+engineering.">Bio-orthogonal, site-selective conjugation of recombinant proteins to microporous annealed particle hydrogels for tissue engineering.</a> Advanced Therapeutics. 3 (1), 1900148 (2020).</li><li>de Rutte, J. M., Koh, J., di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+high-throughput+production+of+modular+microgels+for+in+situ+assembly+of+microporous+tissue+scaffolds.">Scalable high-throughput production of modular microgels for in situ assembly of microporous tissue scaffolds.</a> Advanced Functional Materials. 29 (25), 1900071 (2019).</li><li>Pfaff, B. N., 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=Selective+and+improved+photoannealing+of+microporous+annealed+particle+(MAP)+scaffolds.">Selective and improved photoannealing of microporous annealed particle (MAP) scaffolds.</a> ACS Biomaterials Science and Engineering. 7 (2), 422-427 (2021).</li><li>Darling, N. J., Hung, Y. S., Sharma, S., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+kinetics+of+thiol-maleimide+Michael-type+addition+gelation+kinetics+for+the+generation+of+homogenous+poly(ethylene+glycol)+hydrogels.">Controlling the kinetics of thiol-maleimide Michael-type addition gelation kinetics for the generation of homogenous poly(ethylene glycol) hydrogels.</a> Biomaterials. 101, 199-206 (2016).</li><li>Sandell, J. L., Zhu, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+in-vivo+optical+properties+of+human+tissues+and+its+impact+on+PDT.">A review of in-vivo optical properties of human tissues and its impact on PDT.</a> Journal of Biophotonics. 4 (11-12), 773-787 (2011).</li></ol>

The authors have no conflicts of interest to disclose.

This protocol describes methods for synthesizing and characterizing microgels, which serve as the building blocks for microporous annealed particle (MAP) scaffolds. This protocol uses a high-throughput microfluidic approach to generate large volumes of uniform microgels, which cannot be achieved with other methods such as flow-focusing microfluidics1,4,7,9 (high monodisperisty, low yield), batch emulsion6,10, and electrospraying5,12 (low monodispersity, high yield). With the methods described herein, monodisperse microgels can be made for use in MAP scaffolds that can be used for a variety of regenerative medicine applications (e.g., cell delivery, wound healing).
A critical step of this protocol is the creation of the PDMS microfluidic devices. If the devices are not made correctly, this could have negative downstream effects on microgel formation and monodispersity. It is important to prevent the introduction of artifacts (i.e., bubbles, dust) into the PDMS before it cures, since this could clog the channels and significantly impact microgel formation. To mitigate this as much as possible, one should use tape to remove any dust, store the devices in a dust-free container, and work in a dust-free hood, if possible. It is also recommended to store the devices at 60 &#176;C for the best results with the surface treatment.
When pouring the PDMS devices, it is important to maintain a uniform thickness that is about equal to or less than the length of the biopsy punch. If the device is too thick, the biopsy punch will be unable to penetrate all the way through. It is also crucial to not tear the PDMS device inlets/outlet while punching with the biopsy punch and/or inserting tubing. A tear in the PDMS device will cause leaking from the inlets/outlet, which can cause loss of the gel precursor solution. If there is leaking in a PDMS device, the best solution is to replace it with a new device as quickly as possible.
When plasma-treating the device, the use of pure oxygen and plasma-treating for 30 s has produced the best results for adhering PDMS to the glass slide. If the device does not bond correctly (i.e., the PDMS can still be lifted from the glass slide after plasma treatment), one should double-check that the plasma treater is operating correctly and that the device and slides have been cleaned thoroughly. It is also important to use the correct silane surface treatment, and, for best results, the PDMS devices should be surface-treated directly before use. Other methods of surface treatment, such as chemical vapor deposition, could also be used.
Another crucial step is using the PDMS microfluidic devices correctly for microgel formation. It is recommended to use a flow rate ratio of at least 2:1 (this protocol uses a 6 mL/h oil flow rate and a 3 mL/h aqueous flow rate), but this can be tuned to achieve the desired microgel size. The pH of the microgel precursor solution is also an important metric to optimize to prevent clogging of the device. Phosphate-buffered saline (PBS) accelerates thiolate formation in Michael-type addition chemistry, and the PBS concentrations used in this protocol yield the best results for microgel gelation in the microfluidic devices. Once the syringe pumps are started, there may be some bubbles in the microfluidic channels, but this should equilibrate after a few minutes. It is recommended to monitor microgel formation with a microscope. If the flow does not look similar as in this video and/or there are a few channels producing large particles, this is likely due to issues with the surface treatment step. The best solution is to replace the device with one that has been freshly surface-treated.
If the microgels appear to be coalescing, this may be due to an insufficient concentration of FluoroSurfactant. The recommended solution is to increase the wt% of the surfactant in the oil phase. However, one limitation to using high concentrations of surfactant is that it can be more difficult to remove during the purification step. It is recommended to use microfluidic devices once only, but the devices can be reused if flushed with Novec oil immediately after use to remove any aqueous solution that could gel in the device and clog the channels. While one microfluidic device can produce a high-throughput volume of microgels (mL/h), this production rate can be scaled by using multiple microfluidic devices in parallel.
The annealing step of MAP scaffold assembly relies on the use of a light-activated photoinitiator of radical polymerization, and the photoinitiator can be selected based on the desired application. For example, LAP photoinitiator has fast annealing times (&#60;30 s) when using long-wave UV light, which has minimal impact on cell viability in vitro14. However, this wavelength is highly absorbed by tissue16 and may not have as high an annealing efficacy in vivo as in vitro.
Eosin Y is another photoinitiator activated by visible wavelengths (505 nm) and has deeper penetration into tissue, which enhances the capability of the MAP scaffold to be annealed beneath tissue. However, the long light exposure times needed for Eosin Y annealing may prolong cell exposure to free radicals and impact cell viability in vitro14. Using these methods for high-throughput generation of highly uniform microgel building blocks will accelerate MAP scaffold-focused research and advance knowledge in the field of injectable porous materials for regenerative medicine.

The MAP scaffold platform is an injectable biomaterial composed entirely of hydrogel microparticles (microgels) that provide cell-scale microporosity when crosslinked together, which allows for degradation-independent cell migration and bulk tissue integration1. Due to its ability to quickly integrate with host tissue and inherently low immunogenicity, the MAP scaffold platform has demonstrated preclinical applicability for a wide variety of regenerative medicine therapies2,3,4,5,6,7,8,9,10, including accelerating dermal wound healing1,3,11, revascularizing brain stroke cavity7, delivering mesenchymal stem cells2, and providing tissue bulking to treat glottic insufficiency6. MAP has also been shown to convey anti-inflammatory effects to host tissue through the recruitment of M2 macrophages3 and can even be tuned to promote a Th2 &#34;tissue repair&#34; immune response8. These favorable properties of the MAP scaffold platform allow it to be expanded to a diverse range of clinical applications.
Previously published methods for generating microgels for MAP scaffold formation have included flow-focusing droplet-microfluidics1,4,7,9, electrospraying5,12, and overhead spinning with batch emulsion6,10. The droplet microfluidic method can produce particles with high monodispersity but uses very slow flow rates that produce low yields of particles (&#181;L/h). Alternatively, the electrospraying and batch emulsion methods can produce a high volume of particles, but with high particle polydispersity. This protocol uses a high-throughput microfluidic method to produce microgels with a monodisperse population, based on work by de Rutte et al13. This method utilizes soft lithography techniques to make a polydimethylsiloxane (PDMS) microfluidic device from a photomask, which is then bonded to a glass slide. The device design relies on step-emulsification to generate a high volume of microgel particles (mL/h). The monodispersity that can be achieved with this method provides superior control of porosity compared to other techniques, since monodisperse microgels can form scaffolds with more uniform pore sizes2.
The methods for synthesizing and characterizing the individual microgels that can act as building blocks for MAP scaffolds are outlined within this manuscript, specifically in terms of creating microgels that consist of a PEG backbone with a maleimide (MAL) group, which readily participates in efficient Michael-type addition with thiol-functionalized crosslinkers for microgel gelation. To decouple microgel gelation from MAP scaffold annealing, this manuscript also describes how to synthesize a published14 custom annealing macromer, MethMAL, which is a heterofunctional methacrylamide/maleimide 4-arm PEG macromer. The methacrylamide functional groups readily participate in free-radical photopolymerization (for microgel annealing), while remaining relatively inert to the conditions that promote Michael-type addition for the MAL functional groups.
Additionally, this manuscript outlines the protocols for creating PDMS microfluidic devices, determining microgel gelation kinetics, and characterizing microgel size. The final part of the manuscript details MAP scaffold annealing, which is when the microgels are transitioned in situ into a bulk scaffold through a secondary, photo-initiated crosslinking step that covalently bonds the surfaces of the microgels together. It is important to note that there are other annealing methods that can be implemented in MAP scaffold systems that do not rely on light-based chemistries, such as enzyme-mediated annealing, as previously described1. Overall, these methods can be used directly or used with different hydrogel formulation chemistries (e.g.,&#160;hyaluronic acid-based) to generate MAP scaffolds for any application.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-aminoethanethiol hydrochloride</td>
			<td>Acros Organics</td>
			<td>AC153770250</td>
			<td>For MethMal Synthesis 
			MW: 113.61 Da</td>
		</tr>
		<tr>
			<td>35 mm plate rotor</td>
			<td>HAAKE</td>
			<td>P35/Ti</td>
			<td>Geometry for HAAKE viscometer</td>
		</tr>
		<tr>
			<td>4-arm PEG-Maleimide (10 kDa)</td>
			<td>NOF AMERICA Corporation</td>
			<td>SUNBRIGHT PTE-100MA</td>
			<td>For microgel precursor solution</td>
		</tr>
		<tr>
			<td>4-arm PEG-Maleimide (20 kDa)</td>
			<td>NOF AMERICA Corporation</td>
			<td>SUNBRIGHT PTE-200MA</td>
			<td>For MethMal Synthesis 
			Molecular weight specific to each batch</td>
		</tr>
		<tr>
			<td>BD Syringe with Luer-Lok Tips</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Disposable plastic syringes</td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Mitex</td>
			<td>MLTX33-31A-P/25</td>
			<td>1.5 mm diameter</td>
		</tr>
		<tr>
			<td>Chloroform-d</td>
			<td>Acros Organics</td>
			<td>AC209561000</td>
			<td>For MethMal Synthesis</td>
		</tr>
		<tr>
			<td>Collimated LED Light Source</td>
			<td>ThorLabs</td>
			<td>M365LP1-C1</td>
			<td>365 nm</td>
		</tr>
		<tr>
			<td>Culture dish (15 cm)</td>
			<td>Corning</td>
			<td>CLS430599</td>
			<td>150 mm x 25 mm</td>
		</tr>
		<tr>
			<td>DMTMM(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride)</td>
			<td>Oakwood Chemical</td>
			<td>151882</td>
			<td>For MethMal Synthesis 
			MW: 276.72 Da</td>
		</tr>
		<tr>
			<td>Fluorosurfactant</td>
			<td>Ran Technologies</td>
			<td>008-Flurosurfactant-5wtH-200G</td>
			<td>5 weight percent of 008-Flurosurfactant in HFE7500</td>
		</tr>
		<tr>
			<td>FreeZone Triad Freeze Dry System</td>
			<td>Labconco</td>
			<td>7400000 Series</td>
			<td>For MethMal Synthesis 
			Lyophilizer</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>Plain glass slides, uncoated</td>
		</tr>
		<tr>
			<td>HAAKE Rheowin viscometer</td>
			<td>HAAKE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td>version 1.8.0_172</td>
		</tr>
		<tr>
			<td>KDS Legato 210 Dual Prong Syringe Pump</td>
			<td>Kd Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED Driver</td>
			<td>ThorLabs</td>
			<td>DC2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
			<td>Sigma-Aldrich</td>
			<td>900889</td>
			<td>Photoinitiator</td>
		</tr>
		<tr>
			<td>Methacrylic Acid</td>
			<td>Sigma Aldrich</td>
			<td>155721</td>
			<td>For MethMal Synthesis 
			MW: 86.09 Da 
			Density: 1.015g/mL</td>
		</tr>
		<tr>
			<td>Microfluidic device SU8-Si master wafer</td>
			<td>FlowJem</td>
			<td>N/A</td>
			<td>Custom-made, with silanization</td>
		</tr>
		<tr>
			<td>MMP-2 degradable crosslinker</td>
			<td>FlowJem</td>
			<td></td>
			<td>Sequence: Ac-GCGPQGIAGQDGCG-NH2</td>
		</tr>
		<tr>
			<td>Needles (25 G, beveled)</td>
			<td>BD</td>
			<td>305122</td>
			<td>Length: 15.88 mm 
			Gauge: 0.5 mm</td>
		</tr>
		<tr>
			<td>Novec 7500</td>
			<td>3M</td>
			<td>7100025016</td>
			<td>Fluorinated oil</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Praxair</td>
			<td>UN1072</td>
			<td>Compressed</td>
		</tr>
		<tr>
			<td>Peek tubing</td>
			<td>Trajan Scientific</td>
			<td>03-350-523</td>
			<td>1/32&#34; Outer Diameter; 0.02&#34; Inner Diameter; 10&#39; Length</td>
		</tr>
		<tr>
			<td>PFOCTS (trichloro(1H,1H,2H,2H-perfluorooctyl)silane)</td>
			<td>Sigma-Aldrich</td>
			<td>448931</td>
			<td>For surface treatment</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher BioReagants</td>
			<td>BP3994</td>
			<td>Diluted to 1x in ultrapure water, pH = 7.4</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>RGD cell adhesive peptide</td>
			<td>WatsonBio Sciences</td>
			<td></td>
			<td>Sequence: Ac-RGDSPGGC-NH2</td>
		</tr>
		<tr>
			<td>Rheowin software</td>
			<td>HAAKE&#160;</td>
			<td></td>
			<td>Software compatible with HAAKE viscometer</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Bard-Parker</td>
			<td>371210</td>
			<td>Size: #10</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Bard-Parker</td>
			<td>371030</td>
			<td>Size: #3</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher BioReagents</td>
			<td>BP358-1</td>
			<td>For MethMal Synthesis 
			MW: 58.44 Da</td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>DOW Chemical</td>
			<td>2065622</td>
			<td>Base and curing agent</td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Fisher Scientific</td>
			<td>O4884-100</td>
			<td>For MethMal Synthesis 
			MW: 101.19 Da 
			Density: 0.73g/mL</td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>AAD04103</td>
			<td>ID: 0.51 mm 
			OD: 1.52 mm</td>
		</tr>
		<tr>
			<td>Varian Inova 500 Spectrometer</td>
			<td>Varian</td>
			<td></td>
			<td>NMR 
			Located in the UVA Biomolecular Magnetic Resonance Facility</td>
		</tr>
	</tbody>
</table>

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.

The aim of this protocol is to outline all the steps necessary for synthesizing microgel building blocks to be used in a MAP scaffold. The MethMAL annealing macromer is highly selective and efficient and is compatible with multiple polymer backbones14. It is important that at least 67%-75% of the 20 kDa PEG-maleimide is modified with methacrylamide functional groups to ensure a high annealing efficiency. Percent modification can be most easily determined by analyzing 1H-NMR spectra peaks, as shown in Figure 1. The gelation kinetics, determined by a viscometer, is an important metric to consider for each gel formulation. This protocol uses a gel precursor solution consisting of a PEG backbone with a MAL group, which efficiently reacts with thiol-functionalized crosslinkers for microgel gelation. However, many hydrogel chemistries can be used to fabricate microgels via the high-throughput microfluidic method described herein. The time to onset of gelation will provide insight into the duration of microfluidic microgel generation. It is recommended to choose a gel precursor pH that can initiate gelation between 30 min (Figure 2) and 2 h.
If the gelation time is too fast, the gel precursor solution will begin to polymerize within the microfluidic device and clog the channels. Additionally, it is important to note that changing thiolated ligand concentrations (e.g., RGD) may have impacts on network formation during gelation and may need to be accounted for by adjusting the formulation. The microfluidic device fabrication steps can be tedious, but representative results of a successfully bonded device are shown in Figure 3. This protocol uses a high-throughput, parallelized, step-emulsification microfluidic device that was adapted from a design by de Rutte et al.13, and the silicon wafer fabrication was outsourced to a microfluidic technology company. However, the steps outlined in this protocol can be used with any device design etched on a SU-8 silicon wafer photomask. It is important to note that the step size of the channels on the photomask must be optimized during device fabrication, since it will impact the size of the microgel particles.
The flow rates for the microfluidic generation of microgels should be optimized for each gel formulation based on factors such as gelation time, desired particle size, and microfluidic device design. If using the high-throughput device, the flow rates for the aqueous phase can go as high as 5 mL/h. Figure 4B shows the setup for high-throughput devices used in this protocol. If the device is running correctly, the microgel formation should look similar to that shown in Figure 4D. Before purification, the microgels will be opaque. After completing the various oil, PBS, and hexane washes, the gel should look clear like the representative image in Figure 5. If incorporating a fluorophore into the microgels, the purified product may have a slight colored tint but should still be close to translucent. After purification and swelling, the microgels should be very uniform in size and have a PDI between 1.00 and 1.05, as shown in Figure 6. Various photoinitiators may be used for photoannealing MAP scaffolds. If using an alternative to LAP, described herein, one must determine the annealing kinetics as previously described14. Additionally, various light sources may be used for photoannealing, as long as the light source corresponds with the photoinitiator. One must be sure to calibrate and focus the light source. The annealing time and light intensity may need to be optimized based on gel formulation and photoinitiator concentration. The annealing method outlined in this protocol can be used for in vitro and in vivo studies. After annealing, the microgels will form a porous scaffold that can be visualized with two-photon microscopy (Figure 7B-C).

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Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

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 ...

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