The authors would like to thank the National Institutes of Health, the National Institutes of Neurological Disorders and Stroke (1R01NS112940, 1R01NS079691, R01NS094599), and the National Institute of Allergy and Infectious Disease (1R01AI152568). This work was performed in part at the Duke University Shared Materials Instrumentation Facility (SMIF), a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), which is supported by the National Science Foundation (award number ECCS-2025064) as part of the National Nanotechnology Coordinated Infrastructure (NNCI). The authors would like to thank the lab&#39;s former post-doc Dr. Lucas Schirmer as well as Ethan Nicklow for their assistance in generating the 3D printed device for cell culture experiments.

1. Microfluidic device fabrication
<ol>
	<li>Soft lithography 
	NOTE: This protocol describes device fabrication of a flow-focusing microfluidic device design from de Wilson et al.9. However, this protocol can be used with any device design on an SU-8 wafer. The wafer can be taped to a Petri dish, and then needs to be silanized to prevent adherence of the PDMS to the wafer features15.
	<ol>
		<li>Mix the polydimethylsiloxane (PDMS) elastomer base wit...

<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>Daly, A. C., Riley, L., Segura, T., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+microparticles+for+biomedical+applications.">Hydrogel microparticles for biomedical applications.</a> Nature Reviews Materials. 5 (1), 20-43 (2020).</li><li>Darling, N. 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=Click+by+click+Microporous+Annealed+Particle+(MAP)+scaffolds.">Click by click Microporous Annealed Particle (MAP) scaffolds.</a> Advanced Healthcare Materials. 9 (10), 1901391 (2020).</li><li>Truong, N. F., 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+annealed+particle+hydrogel+stiffness,+void+space+size,+and+adhesion+properties+impact+cell+proliferation,+cell+spreading,+and+gene+transfer.">Microporous annealed particle hydrogel stiffness, void space size, and adhesion properties impact cell proliferation, cell spreading, and gene transfer.</a> Acta Biomaterialia. 94, 160-172 (2020).</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 &amp; Engineering. 7 (2), 422-427 (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 &amp; Engineering. 2 (11), 2034-2041 (2016).</li><li>Caldwell, A. S., Campbell, G. T., Shekiro, K. M. T., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clickable+microgel+scaffolds+as+platforms+for+3D+cell+encapsulation.">Clickable microgel scaffolds as platforms for 3D cell encapsulation.</a> Advanced Healthcare Materials. 6 (15), 1700254 (2017).</li><li>Qazi, T. H., 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=Anisotropic+rod-shaped+particles+influence+injectable+granular+hydrogel+properties+and+cell+invasion.">Anisotropic rod-shaped particles influence injectable granular hydrogel properties and cell invasion.</a> Advanced Materials. 34 (12), 2109194 (2022).</li><li>Wilson, K. 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=Stoichiometric+post+modification+of+hydrogel+microparticles+dictates+neural+stem+cell+fate+in+microporous+annealed+particle+scaffolds.">Stoichiometric post modification of hydrogel microparticles dictates neural stem cell fate in microporous annealed particle scaffolds.</a> Advanced Materials. 34 (33), 2201921 (2022).</li><li>Muir, V. G., Qazi, T. H., Shan, J., Groll, J., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+microgel+fabrication+technique+on+granular+hydrogel+properties.">Influence of microgel fabrication technique on granular hydrogel properties.</a> ACS Biomaterials Science &amp; Engineering. 7 (9), 4269-4281 (2021).</li><li>Highley, C. B., Song, K. H., Daly, A. C., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jammed+microgel+inks+for+3D+printing+applications.">Jammed microgel inks for 3D printing applications.</a> Advanced Science. 6 (1), 1801076 (2018).</li><li>Anderson, A. R., Nicklow, E., 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=Particle+fraction+as+a+bioactive+cue+in+granular+scaffolds.">Particle fraction as a bioactive cue in granular scaffolds.</a> Acta Biomaterialia. 150, 111-127 (2022).</li><li>Pruett, L., Ellis, R., McDermott, M., Roosa, C., 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=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>Sheikhi, 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=Microengineered+emulsion-to-powder+technology+for+the+high-fidelity+preservation+of+molecular,+colloidal,+and+bulk+properties+of+hydrogel+suspensions.">Microengineered emulsion-to-powder technology for the high-fidelity preservation of molecular, colloidal, and bulk properties of hydrogel suspensions.</a> ACS Applied Polymer Materials. 1 (8), 1935-1941 (2019).</li><li>Brower, K., White, A. K., Fordyce, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+variable+height+photolithography+for+valved+multilayer+microfluidic+devices.">Multi-step variable height photolithography for valved multilayer microfluidic devices.</a> Journal of Visualized Experiments. (119), e55276 (2017).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Magnetic+Resonance+(NMR)+Spectroscopy.">Nuclear Magnetic Resonance (NMR) Spectroscopy.</a> JoVE Science Education Database. Organic Chemistry. JoVE. , (2022).</li><li>Roosa, 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=Microfluidic+synthesis+of+microgel+building+blocks+for+microporous+annealed+particle+scaffold.">Microfluidic synthesis of microgel building blocks for microporous annealed particle scaffold.</a> Journal of Visualized Experiments. (184), e64119 (2022).</li><li>Zhang, H., Dicker, K. T., Xu, X., Jia, X., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+bioorthogonal+crosslinking.">Interfacial bioorthogonal crosslinking.</a> ACS Macro Letters. 3 (8), 727-731 (2014).</li><li>Welzel, P. B., 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=Cryogel+micromechanics+unraveled+by+atomic+force+microscopy-based+nanoindentation.">Cryogel micromechanics unraveled by atomic force microscopy-based nanoindentation.</a> Advanced Healthcare Materials. 3 (11), 1849-1853 (2014).</li><li>Plieva, F., Huiting, X., Galaev, I. Y., Bergenst&#229;hl, B., Mattiasson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroporous+elastic+polyacrylamide+gels+prepared+at+subzero+temperatures:+control+of+porous+structure.">Macroporous elastic polyacrylamide gels prepared at subzero temperatures: control of porous structure.</a> Journal of Materials Chemistry. 16 (41), 4065-4073 (2006).</li><li>Rommel, D., 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=Functionalized+microgel+rods+interlinked+into+soft+macroporous+structures+for+3D+cell+culture.">Functionalized microgel rods interlinked into soft macroporous structures for 3D cell culture.</a> Advanced Science. 9 (10), 2103554 (2022).</li><li>Kurt, E., 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=Nucleic+acid+delivery+from+granular+hydrogels.">Nucleic acid delivery from granular hydrogels.</a> Advanced Healthcare Materials. 11 (3), 2101867 (2021).</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 &amp; Engineering. 5 (12), 6395-6404 (2019).</li><li>Truong, N. F., Lesher-P&#233;rez, S. C., Kurt, E., 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=Pathways+governing+polyethylenimine+polyplex+transfection+in+Microporous+Annealed+Particle+scaffolds.">Pathways governing polyethylenimine polyplex transfection in Microporous Annealed Particle scaffolds.</a> Bioconjugate Chemistry. 30 (2), 476-486 (2019).</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>Li, F., 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=Cartilage+tissue+formation+through+assembly+of+microgels+containing+mesenchymal+stem+cells.">Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells.</a> Acta Biomaterialia. 77, 48-62 (2018).</li></ol>

Microfluidic production of HA-NB microgels has been shown to generate microgels with a narrower range of size distribution than emulsion batch production3,9. The microgels described in this protocol were formulated using an MMP-cleavable crosslinker (Ac-GCRDGPQGIWGQDRCG-NH2) to support material degradation.&#160;However, HA-NB microgels can also be crosslinked using an alternative di-thiol linker such as dithiothreitol (DTT), which is non-degradable. S...

Microporous annealed particle (MAP)&#160;scaffolds are a subclass of granular materials in which the microgel (&#181;gel) building blocks are interlinked to form a bulk, porous scaffold. With the unique microarchitecture of these granular scaffolds, the innate porosity generated by the void space between interlinked spherical microgel supports accelerated cell infiltration and migration1. The microgel building blocks of MAP scaffolds can be fabricated from both synthetic and natural polymers with chemical modifications2. The methods described here specifically highlight the use of microgels comprised of a hyaluronic acid (HA) backbone modified with functional norbornene (NB) handles. The NB functional handle on the HA polymer supports click chemistry reactions for forming microgels and linking them together to generate MAP scaffolds3,4. Numerous schemes have been employed for linking the microgels together (i.e., annealing), such as enzymatic1, light-based5,6, and additive-free click chemistry3,7 reactions. Additive-free click chemistry is described in this work, using the tetrazine-norbornene inverse electron demand Diels-Alder conjugation for interlinking the HA-NB microgels.
To fabricate MAP scaffolds, users first generate the microgel building blocks using reverse emulsions either in batch systems or within microfluidic devices, as well as with electrohydrodynamic spraying, lithography, or mechanical fragmentation2. The production of spherical HA-NB microgels has been well described and previously reported using both batch emulsion2 and microfluidic droplet generation techniques8,9,10,11. In this work, spherical HA-NB microgels were generated on a flow-focusing microfluidic platform for controlled size and shape, as previously described8,9,10. After purification, the microgels exist in an aqueous suspension and must be concentrated to induce a jammed state. When jammed, microgels exhibit shear-thinning properties, which allow them to function as injectable, space-filling materials1. One method of inducing a jammed state is to dry the microgels via lyophilization, or freeze-drying, then subsequently rehydrate the dried product in a controlled volume12. Alternatively, excess buffer can be removed from the&#160;microgel slurry via centrifugation over a strainer or with manual removal of the buffer from the microgel pellet either by aspiration or using an absorbent material. However, using centrifugation to dry the microgels can generate a highly variable range of particle fractions and void fractions when making granular scaffolds12. Techniques for lyophilizing microgels have been described using 70% IPA for polyethylene glycol (PEG) microgels13, fluorinated oils for gelatin methacryloyl (GelMa) microgels14, and 70% ethanol for HA microgels12. This protocol highlights methods for freeze-drying spherical HA microgels using 70% ethanol, a standard laboratory reagent, to retain the original microgel properties during the drying process. The freeze-dried HA microgels can be weighed and rehydrated at user-defined weight percentages to control the final particle fractions in MAP scaffolds12.
The final step in MAP scaffold formation relies on annealing the microgels to create a bulk, porous scaffold1. By utilizing native extracellular matrix components and employing bio-orthogonal annealing schemes, MAP scaffolds serve as a biocompatible platform for both in vitro cell culture and in vivo tissue repair3. Through these approaches, MAP scaffolds can be fabricated from HA-NB building blocks with user-defined particle fractions for their employment in tissue engineering applications12. The following protocol describes the microfluidic production of HA-NB microgels followed by lyophilization and rehydration for controlling particle fraction in MAP scaffolds. Lastly, steps for annealing the microgels are described using bio-orthogonal chemistry for in vitro 3D cell culture experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Luer-Lok syringe sterile, single use, polycarbonate</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Luer-Lok syringe sterile, single use, polycarbonate</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 C5 maleimide</td>
			<td>Invitrogen</td>
			<td>A10254</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td>For staining cell culture samples</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>VWR</td>
			<td>89107-726</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch with plunger, 1.0 mm</td>
			<td>Integra Miltex</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch, 4 mm</td>
			<td>Integra Miltex</td>
			<td>33-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt needle, 23 G 0.5&#34;, Non-Sterile, Capped</td>
			<td>SAI Infusion Technologies</td>
			<td>B23-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle-top vacuum filter, 0.22 &#956;m</td>
			<td>Corning</td>
			<td>CLS430521</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>VWR</td>
			<td>1B1110</td>
			<td>For microgel washing buffer</td>
		</tr>
		<tr>
			<td>Capillary-piston assemblies for positive-displacement pipettes, 1000 &#956;L max. volume</td>
			<td>Rainin</td>
			<td>17008609</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary-piston assemblies for positive-displacement pipettes, 25 &#956;L max. volume</td>
			<td>Rainin</td>
			<td>17008605</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary-piston assemblies for positive-displacement pipettes, 250 &#956;L max. volume</td>
			<td>Rainin</td>
			<td>17008608</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube, 15 mL</td>
			<td>CELLTREAT</td>
			<td>667015B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube, 50 mL</td>
			<td>CELLTREAT</td>
			<td>229421</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform, ACS grade, Glass Bottle</td>
			<td>Stellar Scientific</td>
			<td>CP-C7304</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>Corona plasma gun, BD-10A High Frequency Generator</td>
			<td>ETP</td>
			<td>11011</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoTube Vials, Polypropylene, Internal Thread with Screw Cap</td>
			<td>Nunc</td>
			<td>368632</td>
			<td></td>
		</tr>
		<tr>
			<td>D1 mouse mesenchymal cells</td>
			<td>ATCC</td>
			<td>CRL-12424</td>
			<td>Example cell line for culture in MAP gels</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>For staining cell culture samples</td>
		</tr>
		<tr>
			<td>Deuterium oxide, 99.9 atom% D</td>
			<td>Sigma-Aldrich</td>
			<td>151882</td>
			<td>For NMR spectroscopy</td>
		</tr>
		<tr>
			<td>Dialysis tubing, regenerated cellulose membrane, 12-14 kDa molecular weight cut-off</td>
			<td>Spectra/Por</td>
			<td>132703</td>
			<td>For purifying HA-NB and HA-Tet</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>VWR</td>
			<td>BDH1121-4LPC</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>277056</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)&#160;</td>
			<td>TCI-Chemicals</td>
			<td>D2919</td>
			<td>For modifying HA</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>R0861</td>
			<td>Non-degradable dithiol linker (substitute for MMP-cleavable peptide)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma-Aldrich</td>
			<td>D6429-500ML</td>
			<td>For D1 cell culture</td>
		</tr>
		<tr>
			<td>EMS Paraformaldehyde, Granular</td>
			<td>VWR</td>
			<td>100504-162</td>
			<td>For making 4% PFA</td>
		</tr>
		<tr>
			<td>Ethanol absolute (200 proof)</td>
			<td>KOPTEC</td>
			<td>89234-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td>For D1 cell culture</td>
		</tr>
		<tr>
			<td>Heating Plate</td>
			<td>Kopf Instruments</td>
			<td>HP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer with coverglass</td>
			<td>Daigger Scientific</td>
			<td>EF16034F</td>
			<td></td>
		</tr>
		<tr>
			<td>2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hyaluronate, 79 kDa average molecular weight, produced in bacteria Streptococcus zooepidemicus, pharmaceutical grade, microbial contamination &#60;100 CFU/g, bacterial endotoxins &#60;0.050 IU/mg</td>
			<td>Contipro</td>
			<td>N/A</td>
			<td>79 kDa average molecular weight was used for HA-Tet synthesis, but these methods could be adapted for other molecular weights.</td>
		</tr>
		<tr>
			<td>IMARIS Essentials software package</td>
			<td>Oxford Instruments</td>
			<td>N/A</td>
			<td>Microscopy image analysis software</td>
		</tr>
		<tr>
			<td>Infusion pump, dual syringe</td>
			<td>Chemyx</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory stand with support lab clamp</td>
			<td>Geyer</td>
			<td>212100</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Airgas</td>
			<td>NI 180LT22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate</td>
			<td>TCI-Chemicals</td>
			<td>L0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td>Labconco FreeZone 6 plus has been discontinued, but other lab grade console freeze dryers could be used for this protocol.</td>
		</tr>
		<tr>
			<td>Methyltetrazine-PEG4-maleimide</td>
			<td>Kerafast</td>
			<td>FCC210</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>2-(4-Morpholino)ethane Sulfonic Acid (MES)</td>
			<td>Fisher Scientific</td>
			<td>BP300-100</td>
			<td>For modifying HA</td>
		</tr>
		<tr>
			<td>Micro cover glass, 24 x 60 mm No. 1</td>
			<td>VWR</td>
			<td>48393-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic device SU8 master wafer</td>
			<td>FlowJem</td>
			<td></td>
			<td>Custom design made either in-house in clean room or outsourced</td>
		</tr>
		<tr>
			<td>Mineral oil, heavy</td>
			<td>Sigma-Aldrich</td>
			<td>330760</td>
			<td></td>
		</tr>
		<tr>
			<td>MMP-cleavable dithiol crosslinker peptide (Ac-GCRDGPQGIWGQDRCG-NH2)</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Norbornene-2-methylamine</td>
			<td>TCI-Chemicals</td>
			<td>95-10-3</td>
			<td>For HA-NB synthesis</td>
		</tr>
		<tr>
			<td>Packing tape</td>
			<td>Scotch</td>
			<td>3M 1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG(thiol)2</td>
			<td>JenKem Technology USA</td>
			<td>A4001-1</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, 10,000 units/mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>For D1 cell culture</td>
		</tr>
		<tr>
			<td>Petri dish, polystyrene, disposable, Dia. x H=150 x 15 mm</td>
			<td>Corning</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td>For washing HMPs</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1x</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>RainX water repellent glass treatment</td>
			<td>Grainger</td>
			<td>465D20</td>
			<td>Synthetic hydrophobic treatment solution for microfluidic device treatment</td>
		</tr>
		<tr>
			<td>RGD peptide (Ac-RGDSPGERCG-NH2)</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td>Staples</td>
			<td>112417</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Chem-Impex</td>
			<td>30070</td>
			<td>For dialysis</td>
		</tr>
		<tr>
			<td>Span 80 for synthesis</td>
			<td>Sigma-Aldrich</td>
			<td>1338-43-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer</td>
			<td>Electron Microscopy Science</td>
			<td>4019862</td>
			<td>polydimethylsiloxane (PDMS) elastomer for making microfluidic devices and tissue culture devices</td>
		</tr>
		<tr>
			<td>Syringe filter, Whatman Uniflo, 0.2 &#956;m PES, 13 mm diameter</td>
			<td>Cytvia</td>
			<td>09-928-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetraview LCD digital microscope</td>
			<td>Celestron</td>
			<td>44347</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazine-amine HCl salt</td>
			<td>Chem-Impex</td>
			<td>35098</td>
			<td>For HA-Tet synthesis</td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>471283</td>
			<td>For synthesis of fluorescently-labeled tetrazine</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td>Millipore Sigma</td>
			<td>51805-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>97063-864</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.25%), Phenol red</td>
			<td>Fisher Scientific</td>
			<td>25-200-056</td>
			<td>For lifting adherent cells to seed in MAP gels</td>
		</tr>
		<tr>
			<td>Tygon ND-100-80 Non-DEHP Medical Tubing, Needle Gauge=23, Wall Thickness=0.020 in, Internal diameter = 0.020, Outer diameter = 0.060 in</td>
			<td>Thomas Scientific</td>
			<td>1204G82</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing system controller, LX500 LED&#160;</td>
			<td>OmniCure</td>
			<td>010-00369R</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing head, LED spot UV</td>
			<td>OmniCure</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light meter, Traceable</td>
			<td>VWR</td>
			<td>61161-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td>Bel-Art</td>
			<td>08-594-15C</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Acto Z Series Precision Utility Knife</td>
			<td>Elmer&#39;s</td>
			<td>XZ3601W</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The aim of this protocol is to demonstrate the preparation of microporous annealed particle (MAP) scaffolds with a bio-orthogonal crosslinking scheme as well as controlled particle fractions for 3D cell culture. First, HA was modified with norbornene pendant groups to be used in both microgel formation and interlinking to form MAP scaffolds. Using these methods, approximately 31% of HA repeat units were successfully modified with a norbornene functional handle (Figure 1A). Microfluidic devic...

Watch this Scientific Journal Video about Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture at JoVE.com

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

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 ...

Granular materials, such as MAP scaffolds, are porous injectable materials made from interlinked micro-size hydrogel particles. Controlling particle fraction is critical to material properties and cellular responses. Variable particle fractions in MAP scaffolds result in a range of scaffold properties and cell responses, but this technique allows users to define the final particle fraction in the MAP scaffolds they create.MAP scaffolds can be used to promote the repair and regeneration of complex wounds, as well to deliver drugs. Particle fraction, for example, affects the rate and degree of cellular infiltration and regeneration. This method can be applied across all classes of granular hydrogels.Now that we can make MAP scaffolds with user-controlled particle fractions, we can reproducibly study the bioactivity of these materials and investigate unique cell responses, such as confinement. Setting up the microfluidic microgel generation requires time and patience as you learn the techniques. It's best to have multiple devices prepared and at the ready in case you run into issues with the setup.Begin with microfluidic production of Hyaluronic Acid, or HA, and Norbornene, or NB, microgels. Prior to drying the microgels, weigh a cryo-safe screw-cap tube. Then in a sterile hood, transfer the washed and purified microgels to a cryo-safe screw-cap tube using a positive displacement pipette.Add 70%ethanol to the purified microgels and mix well. Centrifuge the tube for five minutes at 5, 000 g. Aspirate the supernatant, and add 70%ethanol.Mix well and incubate overnight at four degrees Celsius. The next day, briefly centrifuge to ensure the microgels are at the bottom of the tube. Add liquid nitrogen to a cryogenic container, and place the tube of microgels to flash freeze.After five to 10 minutes, remove the tube of microgels with forceps. Quickly remove the cap, and cover the tube with lab-grade tissue. Secure the tissue with a rubber band, and transfer it to a lyophilization container, or chamber.Load the sample on the lyophilizer following the manufacturer's instructions, and lyophilize at 0.066 Torr and minus 63 degrees Celsius. Weigh the lyophilized microgels from the tube before storing them at room temperature. Mix polydimethylsiloxane, or PDMS elastomer base with the curing agent at a 10 to one ratio by mass.Pour the PDMS mixture into a large plastic Petri dish and degas in the desiccator for 30 minutes, or until all the bubbles have disappeared. Once all the bubbles have disappeared, carefully place the 3D-printed mold into the PDMS to minimize the formation of new bubbles. Place in the oven at 60 degrees Celsius for two hours to cure the PDMS.After curing, use a knife, or razor blade to gently trace around the perimeter of the culture device, and carefully remove the mold. Use a four-millimeter biopsy punch to remove any PDMS from the bottom of the wells. Cut the devices to fit on a glass cover slip.Use tape to remove dust from the bottom side of the culture device. Place the clean glass cover slip and culture device on a hot plate at 135 degrees Celsius for 15 minutes to remove moisture. In a fume hood, use a corona plasma gun high on the glass cover slip and the bottom side of the device for 30 seconds.Then quickly bond the treated surfaces together. Gently apply pressure to ensure a good seal between the culture device and glass cover slip. Place the device in a 60 degree Celsius oven overnight to secure the bond.The following day, autoclave the device to sterilize before use in vitro. Next, prepare the Microporous Annealed Particle, or MAP scaffold components, based on the desired particle fraction for cell culture in MAP scaffolds. Weigh lyophilized microgels to determine the mass of microgels.Then reconstitute the microgels in 84%of the final MAP volume of cell media based on the chosen weight percent MAP. Allow the microgels to swell for 20 minutes. Dissolve the Hyaluronic Acid, or HA-tetrazine, and cell media in 16%of the final MAP volume.Once cells have reached the desired confluency, lift and count the cells. Transfer 10, 000 cells per microliter MAP to a new tube. Centrifuge the cells, and aspirate the supernatant from the pellet without aspirating the cells.Add microgels and cross-linker to the pellet using a displacement pipette. Mix well, and seed 10 microliters per well. Pipette in a circular motion to evenly distribute the mixture in the well.Allow the microgels to anneal at 37 degrees Celsius for 25 minutes before adding cell media to fill the wells. Maintain the 3D cultures at 37 degrees Celsius, and change media as needed. To avoid aspirating the scaffold, stabilize the pipette tip along the ridge of the upper well.At the desired time points, fix samples by replacing media with 50 microliters of 4%paraformaldehyde per well for 30 minutes at room temperature. Wash the samples three times with 50 microliters of PBS, or preferred buffer, and proceed for immunofluorescence, or fluorescence staining using 50 microliters per well as the working volume. Microfluidic devices with a flow-focusing region were shown to produce HA-NB microgels of either 50, or 100 microns in diameter.The medium for lyophilizing the microgels was optimized to minimize cryogel formation by using 70%ethanol. However, other media, such as isopropyl alcohol, water, and acetonitrile can be used interchangeably to facilitate cryogel formation. To facilitate bio-orthogonal interlinking of HA-NB microgels a linear HA-tetrazine cross-linker was synthesized.Proton NMR spectroscopy shows a successful modification of 11%of HA repeat units with a tetrazine pendant group. The dried lyophilized microgel product was rehydrated with special volumes and annealed to achieve different weight percent formulations of microgels in MAP scaffolds. These weight percentages of microgels in MAP scaffolds corresponded to unique particle fractions.Cell culture in MAP scaffolds is shown here. Cells in 3D culture in MAP scaffolds were fixed on day five, stained and imaged on a confocal microscope. Single Z-slices show differences in cell growth and scaffolds comprising different weight percent MAP.Select your lyophilization media appropriately depending on whether you want cryogels, or not. Do not skip incubation steps, so that your microgels have sufficient time to swell in the media. Standard techniques such as microscopy, flow cytometry, RNA sequencing, and more can be used to assess cell responses within these granular materials with defined porosities.

controlling-particle-fraction-microporous-annealed-particle-scaffolds

Research

JoVE Journal

Bioengineering

2.1K visualizações.  Duke University. Materiais granulares, como andaimes MAP, são materiais injetáveis porosos feitos de partículas de hidrogel de tamanho micro interligadas. O controle da fração de partículas é fundamental para as propriedades do material e as respostas celulares. Frações de partículas variáveis em andaimes MAP resultam em uma variedade de propriedades de andaimes e respostas celulares, mas essa técnica permite que os usuários definam a fração de partícula final nos andaimes MAP que eles criam.