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).

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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3.0K Views.  University of Virginia. 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.

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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic Mixers for Studying Protein Folding

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

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

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

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

Summary

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Chapters in this video

Fluorescence detection methods for microfluidic droplet platforms

Microfluidic Mixers for Studying Protein Folding

A Microfluidic Chip for ICPMS Sample Introduction

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

A Protocol for Real-time 3D Single Particle Tracking

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Microfluidic Model to Mimic Initial Event of Neovascularization