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/pubmed?term=((Accelerated+wound+healing+by+injectable+microporous+gel+scaffolds+assembled+from+annealed+building+blocks%5BTitle%5D)+AND+%22Nature+Materials%22%5BJournal%5D)">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/pubmed?term=((Enhanced+in+vivo+delivery+of+stem+cells+using+microporous+annealed+particle+scaffolds%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">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/pubmed?term=((Heparin+microislands+in+microporous+annealed+particle+scaffolds+for+accelerated+diabetic+wound+healing%5BTitle%5D)+AND+%22Advanced+Functional+Materials%22%5BJournal%5D)">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/pubmed?term=((Injectable+and+spatially+patterned+microporous+annealed+particle+%28MAP%29+hydrogels+for+tissue+repair+applications%5BTitle%5D)+AND+%22Advanced+Science%22%5BJournal%5D)">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/pubmed?term=((Microporous+bio-orthogonally+annealed+particle+hydrogels+for+tissue+engineering+and+regenerative+medicine%5BTitle%5D)+AND+%22ACS+Biomaterials+Science+and+Engineering%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+microporous+annealed+particle+hydrogel+for+long-term+vocal+fold+augmentation%5BTitle%5D)+AND+%22Laryngoscope%22%5BJournal%5D)">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/pubmed?term=((Injection+of+microporous+annealing+particle+%28MAP%29+hydrogels+in+the+stroke+cavity+reduces+gliosis+and+inflammation+and+promotes+NPC+migration+to+the+lesion%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">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/pubmed?term=((Activating+an+adaptive+immune+response+from+a+hydrogel+scaffold+imparts+regenerative+wound+healing%5BTitle%5D)+AND+%22Nature+Materials%22%5BJournal%5D)">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/pubmed?term=((Particle+hydrogels+based+on+hyaluronic+acid+building+blocks%5BTitle%5D)+AND+%22ACS+Biomaterials+Science+and+Engineering%22%5BJournal%5D)">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/pubmed?term=((Injectable+microannealed+porous+scaffold+for+articular+cartilage+regeneration%5BTitle%5D)+AND+%22Annals+of+Plastic+Surgery%22%5BJournal%5D)">Injectable microannealed porous scaffold for articular cartilage regeneration.</a> Annals of Plastic Surgery. 84, 6S Suppl 5 446-450 (2020).</li>
<li>Pruett, L., Ellis, R., McDermott, M., Roosa, C., Griffin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatially+heterogeneous+epidermal+growth+factor+release+from+microporous+annealed+particle+%28MAP%29+hydrogel+for+improved+wound+closure%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry+B%22%5BJournal%5D)">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/pubmed?term=((Bio-orthogonal%2C+site-selective+conjugation+of+recombinant+proteins+to+microporous+annealed+particle+hydrogels+for+tissue+engineering%5BTitle%5D)+AND+%22Advanced+Therapeutics%22%5BJournal%5D)">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/pubmed?term=((Scalable+high-throughput+production+of+modular+microgels+for+in+situ+assembly+of+microporous+tissue+scaffolds%5BTitle%5D)+AND+%22Advanced+Functional+Materials%22%5BJournal%5D)">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/pubmed?term=((Selective+and+improved+photoannealing+of+microporous+annealed+particle+%28MAP%29+scaffolds%5BTitle%5D)+AND+%22ACS+Biomaterials+Science+and+Engineering%22%5BJournal%5D)">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/pubmed?term=((Controlling+the+kinetics+of+thiol-maleimide+Michael-type+addition+gelation+kinetics+for+the+generation+of+homogenous+poly%28ethylene+glycol%29+hydrogels%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((A+review+of+in-vivo+optical+properties+of+human+tissues+and+its+impact+on+PDT%5BTitle%5D)+AND+%22Journal+of+Biophotonics%22%5BJournal%5D)">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><tbody><tr><td>2-aminoethanethiol hydrochloride</td><td>Acros Organics</td><td>AC153770250</td><td>For MethMal Synthesis&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&amp;nbsp;</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&lt;br/&gt; MW: 86.09 Da&lt;br/&gt; 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-NH&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Needles (25 G, beveled)</td><td>BD</td><td>305122</td><td>Length: 15.88 mm&lt;br/&gt; 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&quot; Outer Diameter; 0.02&quot; Inner Diameter; 10&#039; 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-NH&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Rheowin software</td><td>HAAKE&amp;nbsp;</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&lt;br/&gt; 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&lt;br/&gt; MW: 101.19 Da&lt;br/&gt; 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&lt;br/&gt; OD: 1.52 mm</td></tr><tr><td>Varian Inova 500 Spectrometer</td><td>Varian</td><td></td><td>NMR&lt;br/&gt; 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).

Watch this Scientific Journal Video about Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold at JoVE.com

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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Bioengineering

3.1K Views.  University of Virginia. De high throughput fabricage van monodispersed microgel bouwstenen voor microporeuze gegloeide deeltjessteigers zorgt voor een verbeterde controle van de porositeit van steigers. Dit kan van invloed zijn op de poriegrootte en de daaropvolgende resultaten van weefselintegratie. Dit protocol maakt gebruik van een microfluïdische benadering met hoge doorvoer om grote hoeveelheden monodispersed microgels te genereren die niet kunnen worden bereikt met andere methoden zoals flowfocusmicrofluïdic...

Video: Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

Chemistry

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

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 ...

Environment

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.), ...

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 ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

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 ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation in situ. Unlike conventional bulk hydrogels, interconnected microscale pores in GHS facilitate degradation-independent cell infiltration as well as oxygen, nutrient, and cellular byproduct transfer. Methacryloyl-modified gelatin (GelMA), a (photo)chemically crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties, has widely been used as a cell-responsive/instructive biomaterial. Converting bulk GelMA to GHS&#160;may open a plethora of opportunities for tissue engineering and regeneration. In this article, we demonstrate the procedures of high-throughput GelMA microgel fabrication, conversion to resuspendable dry microgels (micro-aerogels), GHS formation via the chemical assembly of microgels, and granular bioink fabrication for extrusion bioprinting. We show how a sequential physicochemical treatment via cooling and photocrosslinking enables the formation of mechanically robust GHS.&#160;When light is inaccessible (e.g., during deep tissue injection), individually crosslinked GelMA HMPs may be bioorthogonally assembled via enzymatic crosslinking using transglutaminases. Finally, three-dimensional (3D) bioprinting of microporous GHS&#160;at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.

This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting.

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

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