We express our gratitude to the coauthors of our previous work this methodology is based on, C&#233;line Bastard, Luis P. B. Guerzoni, Yonca Kittel, Rostislav Vinokur, Nikolai Born, and Tam&#225;s Haraszti. We gratefully acknowledge funding from the Deutsche Forschungsgemeinschaft (DFG) within the project B5 and C3 SFB 985 &#34;Functional Microgels and Microgel Systems&#34;. We acknowledge funding from the Leibniz Senate Competition Committee (SAW) under the Professorinnenprogramm (SAW-2017-PB62: BioMat). We sincerely acknowledge funding from the European Commission (EUSMI, 731019). This work was performed in part at the Center for Chemical Polymer Technology (CPT), which was supported by the EU and the federal state of North Rhine-Westphalia (grant EFRE 30 00 883 02).

1. Required material and preparations for microfluidics
<ol>
	<li>For the described microfluidic procedure, use 1 mL and 5 mL glass syringes and syringe pumps. On-chip droplet formation is observed via an inverted microscope equipped with a high-speed camera.</li>
	<li>Create the microfluidic chip design (Figure 1B) using a computer-aided design software and produce a master template as already reported12.</li>
	<li>Achieve controlled UV-irradiation using a self-constructed UV-LED (&#955; = 365 nm, spot diameter ~4.7 mm) providing irradiance at 957 mW/cm2 through a 0.13 mm thick cover glass. 
	NOTE: Take into account possible local heat generation by the UV-LED during irradiation. If this is the case, ensure sufficient cooling by external airflow.</li>
</ol>
2. Microfluidic device production
NOTE: The microfluidic device production is based on a previous publication13.
<ol>
	<li>Prepare 20 g of a 10:1 (by mass) mixture of polydimethylsiloxane (PDMS) and curing agent. Mix vigorously for 3 min.</li>
	<li>Mix 60 mg of Oil Red O in 2.0 g of toluene. Add 50 &#181;L of Oil Red O in toluene solution to the PDMS mixture. Mix until there is homogeneous color distribution to decrease undesired light scattering and keep the spot size focused during on-chip gelation.</li>
	<li>Remove the bubbles by placing the mixture into a desiccator equipped with a vacuum pump.</li>
	<li>Cast the PDMS mixture into the master template to a height of 5-5.5 mm and degas again.</li>
	<li>Allow the PDMS to cure for 18 h at room temperature to avoid diazo dye degradation.</li>
	<li>Cut out the PDMS structure and punch inlet and outlet holes into the structure using a line core sampling tool (0.77 mm inner diameter, 1.07 mm outer diameter).</li>
	<li>Wash the PDMS and cover glass with isopropanol and deionized water repeatedly 5x and, subsequently, remove the liquid via pressured air or nitrogen flow after each washing step.</li>
	<li>Treat the dry glass and PDMS together in a plasma oven at 0.2 mbar, with an oxygen flow of 20 mL/min for 60 s at 100 W, and connect directly to bind the glass and PDMS together to form the microfluidic device. 
	NOTE: Avoid bending the PDMS structure during the binding process to minimize channel structure deformation.</li>
	<li>To render the channels of the microfluidic chip hydrophobic, place the device together with 50 &#181;L of trichloro-(1H,1H,2H,2H-perfluoroctyl)-silane in a desiccator under vacuum overnight (close the connection to the pump after decreasing the vapor pressure). Rinse the outside of the device with hydrofluoroether. 
	CAUTION: Perform these steps in a fume hood and avoid any contact with the perfluoro silane. Use a glass vacuum desiccator sealed with grease. Clean the desiccator thoroughly before using for other purposes.</li>
</ol>
3. Solution preparation for microfluidics
<ol>
	<li>For the continuous oil phase, mix paraffin oil and hexadecane (1:1 v/v) and add 8% (w/w) of a non-ionic surfactant. Fill one 1 mL (first oil, Figure 1) and one 5 mL (second oil, Figure 1) glass syringe.</li>
	<li>Prepare the prepolymer solutions for the disperse phase in brown glass vials to prevent photoinitiator decomposition and unintended gelation.
	<ol>
		<li>Amine component prepolymer solution containing GRGDS-PC
		<ol>
			<li>For a 300 &#181;L solution, weigh out 33.3 mg of star-polyethylene glycol-acrylate (sPEG-AC) and 3.03 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).</li>
			<li>Dissolve 18.74 mg of 2-aminoethyl methacrylate hydrochloride (AMA) in 1.5 mL of deionized water and pass the solution through a syringe filter (0.20 &#181;m pore size). 
			NOTE: AMA is hygroscopic and should be discarded as soon as the solid forms lumps in the storage container.</li>
			<li>Prepare sterile 36 mM GRGDS-PC aliquots in water and keep at &#8722;20 &#176;C until use.</li>
			<li>Use 291.7 &#181;L of the AMA solution to dissolve sPEG-AC and LAP, and add 8.3 &#181;L of the GRGDS-PC solution. Take up the solution with a 1 mL glass syringe and protect from light using aluminum foil.</li>
		</ol>
		</li>
		<li>Epoxy component prepolymer solution
		<ol>
			<li>For a 300 &#181;L solution, weigh 33.3 mg of star-polyethylene glycol-acrylate (sPEG-AC), 3.03 mg of LAP and dissolve in 300 &#181;L of deionized water. Add 2.4 mg of glycidyl methacrylate (GMA). 
			NOTE: Vigorous shaking accelerates GMA dissolving. Take up the solution with a 1 mL glass syringe and protect from light using aluminum foil.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Production and purification of amine and epoxy functionalized microgel rods
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64010/64010fig01v2.jpg" /> 
Figure 1: Arrangement of the microfluidic on-chip gelation assembly. (A) Front view and angled view of the component arrangement during microfluidics. (B) Microfluidic chip design used for on-chip gelation of microgel rods. (1) PE tube to the first oil inlet. (2) Light-protected PE tube to the disperse phase inlet. (3) PE tube to the second oil inlet. (4) PE tube from the outlet to the product collection container. (5) UV lamp and irradiation location on the straight 80 &#181;m channel near the outlet. (6) Microscope objective/observation position. (7) Colored PDMS component of the microfluidic device. (8) Cover glass bonded to the PDMS. <a href="https://www.jove.com/files/ftp_upload/64010/64010fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Insert the needles into the PE tubes and remove the gas from the syringe and the tube.</li>
	<li>Insert a PE tube into the outlet for product collection.</li>
	<li>Place all the glass syringes in the syringe pumps and insert each tubing end into the corresponding inlet (Figure 1). 
	NOTE: Protect the prepolymer tubing from light via aluminum foil or a black tube to avoid unintentional gelation.</li>
	<li>Focus the microscope on the oil-water cross-section.</li>
	<li>Start the first oil syringe pump (flow rate = 100-200 &#181;L/h) to fill the channel with oil first to prevent channel wetting by the disperse phase.</li>
	<li>Decrease the first oil flow rate to 30 &#181;L/h and start the prepolymer syringe pump (flow rate = 100-200 &#181;L/h) until the dispersed aqueous phase can be observed at the cross-section.</li>
	<li>Set the prepolymer flow rate to 30 &#181;L/h and focus the microscope on the outlet.</li>
	<li>Start the second oil syringe pump (flow rate of 300 &#181;L/h) and wait until the flow regime is stable.</li>
	<li>Place the outlet tube in a collection container. 
	NOTE: Place the end of the tube in the upper part of the container to avoid a pressure increase due to the accumulation of the product over time.</li>
	<li>Set the UV irradiation system such that the irradiance is in the range of 900-1000 mW/cm2&#160;(used irradiance = 957 mW/cm2) and the irradiation spot is in the straight channel part before the outlet (Figure 1B). 
	NOTE: Make sure to not irradiate near the oil-water cross-section to avoid clogging the channel. For additional protection, cover the channel structures prior to the irradiation spot on the top of the unit.</li>
	<li>Before UV irradiation, adjust the flow rates of the prepolymer and the first oil to achieve the desired aspect ratio in the range of 3.0 to 4.5, and set the irradiation time of the dispersed phase to ~2.3 s, depending on the size of the irradiation spot.</li>
	<li>Start UV irradiation and, if necessary, adjust the flow rates again according to the previous subsection. 
	NOTE: Observe the production at the beginning and monitor for stable flow behavior in the outlet and the uniformity of the microgel rods. The second oil flow can be adjusted to optimize product transport within the outlet.</li>
	<li>Change the collection container and note the product collection start time and flow rates. 
	NOTE: Protect the product from dust. Stop collecting before any syringe runs low on solution.</li>
	<li>To end collection, remove the collection container, noting the time. Stop irradiation and all the syringe pumps.</li>
	<li>Wash the product subsequently 5x each with n-hexane, isopropanol, and deionized water. Remove the supernatant after rod sedimentation. 
	NOTE: After each solution addition, disperse the product carefully and wait 10 min before replacing the solvent, considering molecular diffusion. Replace the isopropanol gradually with water to prevent the rods from floating to the surface of the liquid. Multiple additional washing steps with water decrease the remaining isopropanol traces.</li>
</ol>
5. Macroporous scaffold formation
<ol>
	<li>Determine the number of microgel rods per dispersion volume for the epoxy and amine functionalized samples.</li>
	<li>Adjust the number of epoxy and amine functionalized samples by dilution or concentration to a similar value between 1,000-5,000 rods/100 &#181;L. 
	NOTE: Use a centrifuge at ~2,000 x g for 5-10 s to speed up the sedimentation of the microgel rods. If the number of rods per dispersion volume is low, rod-interlinking may result in smaller rod clusters instead of one stable construct. If the number of rods per dispersion volume is high, the mixing quality of the two components will be compromised.</li>
	<li>Transfer the first component (~1,200 rods) dispersion into a conical 1.5 mL or 2 mL transparent vial.</li>
	<li>Add the second component in a controlled manner in a continuous operation (100 &#181;L pipette). After addition, mix the contents directly using the pipette to take up liquid and add it again. The interlinked structure forms within seconds during mixing. 
	NOTE: If multiple clusters form instead of one coherent structure, recheck the number of microgel rods per volume or provide more controlled mixing of the two components.</li>
</ol>
6. Cell adhesive post-modification
<ol>
	<li>Calculate the theoretical number of epoxy groups in the interlinked structure based on the flow rate of the dispersed polymer phase, the number of microgels collected during a specific time, and the dilution factor of the microgel dispersion used for scaffold formation. 
	NOTE: Approximate the theoretical number of epoxy groups per scaffold by the following equation. 
	<img alt="Equation 1" src="/files/ftp_upload/64010/64010eq01v3.jpg" style="margin: auto;" /> 
	<img alt="Equation 2" src="/files/ftp_upload/64010/64010eq02v2.jpg" style="margin: auto;" /> 
	nth: Theoretical amount of substance 
	cGMA: GMA concentration in prepolymer solution 
	Q: Flow rate of prepolymer solution</li>
	<li>Add GRGDS-PC solution to the interlinked structure to modify all remaining epoxy groups with the cell adhesive peptide bearing a free amine and thiol (n[theoretical epoxy groups]/n[GRGDS-PC] = 1). Leave at room temperature overnight.</li>
	<li>Remove the unreacted molecules by washing with deionized water and removing the supernatant.</li>
</ol>
7. Sterilization and transfer into cell culture media
<ol>
	<li>Reduce the water level to just submerge the interlinked scaffold.</li>
	<li>Open the vial and irradiate with UV light of &#955; = 250-300 nm. Close the vial and transfer the vial onto a clean bench. Wash 1x with sterile water.</li>
	<li>Replace the water in the vial with cell culture media and allow for equilibration for 5 min. Repeat this with fresh cell culture media 2x.</li>
	<li>Transfer the macroporous scaffold into a cell culture well plate for the experiment by pouring or using a spatula.</li>
</ol>
&#160;&#160;

<ol>
	<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>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>Xin, S., Wyman, O. M., 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=Assembly+of+PEG+microgels+into+porous+cell-instructive+3D+scaffolds+via+thiol-ene+click+chemistry.">Assembly of PEG microgels into porous cell-instructive 3D scaffolds via thiol-ene click chemistry.</a> Advanced Healthcare Materials. 7 (11), 1800160 (2018).</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 (2019).</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=Microfluidic-enabled+bottom-up+hydrogels+from+annealable+naturally-derived+protein+microbeads.">Microfluidic-enabled bottom-up hydrogels from annealable naturally-derived protein microbeads.</a> Biomaterials. 192, 560-568 (2019).</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>Hsu, R. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptable+microporous+hydrogels+of+propagating+NGF-gradient+by+injectable+building+blocks+for+accelerated+axonal+outgrowth.">Adaptable microporous hydrogels of propagating NGF-gradient by injectable building blocks for accelerated axonal outgrowth.</a> Advanced Science. 6 (16), 1900520 (2019).</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>Chen, Z., 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=Advanced+microfluidic+devices+for+fabricating+multi-structural+hydrogel+microsphere.">Advanced microfluidic devices for fabricating multi-structural hydrogel microsphere.</a> Exploration. 1 (3), 20210036 (2021).</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>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>Guerzoni, L. 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=Cell+encapsulation+in+soft,+anisometric+poly(ethylene)+glycol+microgels+using+a+novel+radical-free+microfluidic+system.">Cell encapsulation in soft, anisometric poly(ethylene) glycol microgels using a novel radical-free microfluidic system.</a> Small. 15 (20), 1900692 (2019).</li><li>Kr&#252;ger, A. J. 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=Compartmentalized+jet+polymerization+as+a+high-resolution+process+to+continuously+produce+anisometric+microgel+rods+with+adjustable+size+and+stiffness.">Compartmentalized jet polymerization as a high-resolution process to continuously produce anisometric microgel rods with adjustable size and stiffness.</a> Advanced Materials. 31 (49), 1903668 (2019).</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>Lutzweiler, G., Ndreu Halili, ., Engin Vrana, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+overview+of+porous,+bioactive+scaffolds+as+instructive+biomaterials+for+tissue+regeneration+and+their+clinical+translation.">The overview of porous, bioactive scaffolds as instructive biomaterials for tissue regeneration and their clinical translation.</a> Pharmaceutics. 12 (7), 602 (2020).</li><li>Dang, H. P., 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=3D+printed+dual+macro-,+microscale+porous+network+as+a+tissue+engineering+scaffold+with+drug+delivering+function.">3D printed dual macro-, microscale porous network as a tissue engineering scaffold with drug delivering function.</a> Biofabrication. 11 (3), 035014 (2019).</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 (2019).</li></ol>

The authors assure that there are no conflicts of interest.

One of the critical steps in this protocol is the quality of the 2-aminoethyl methacrylate (AMA) used as the comonomer for primary amine functionalization. The AMA should be a fine-grained and preferably colorless powder delivered in a gas-tight brown glass container. One should avoid using greenish and lumpy material, as it significantly impairs the gelation reaction and negatively affects the reproducibility of the results. In case of poor gelation and unstable microgel rods, one can consider changing the supplier....

To study complex cooperative cell behavior in 3D constructs, scaffold platforms need to show consistent performance in reproducibility, have suitable geometry for cell migration, and, at the same time, allow certain flexibility in terms of parameter alteration to investigate their influence on the living tissue1. In recent years, the concept of macroporous annealed particles (MAP), first described by Segura et al., developed into an efficient and versatile platform for 3D scaffold production2. The tailored composition of the microgels, which are the building blocks of the final 3D scaffold, predefines properties such as the stiffness of the construct, the selective chemical reactivity of the gel network, and the final pore size of the scaffold2,3,4,5,6. Cell adhesive peptides as cues for scaffold-cell interactions are incorporated into the polymer network of the microgels to allow for cell attachment and can be varied to investigate their specific effects on cells in culture. The 3D scaffolds are stabilized by interlinking of the annealed injectable microgels due to covalent or supramolecular bonds, resulting in robust and defined constructs for cell culture2,3,5,7,8.
Microfluidics has established itself as one of the most accurate and adaptable methods for the preparation of defined granular hydrogels9. The possibility of producing larger quantities of the required building blocks in a continuous process while maintaining their chemical, mechanical, and physical monodispersity contributes substantially to the suitability of this process. Furthermore, the size and shape of the produced microgels can be manipulated by various methods such as batch emulsions, microfluidics, lithography, electrodynamic spraying, or mechanical fragmentation, which determine the geometry of the building blocks and, thus, the 3D structure of the final scaffold1,10.
Recently, the concept of macroporous 3D scaffolds composed of functionalized microgel rods that rapidly interlink in aqueous solutions without further additives has been reported11. The anisotropy of microgel rods resulted in higher porosities and pore distributions with larger pore sizes compared to employing spherical microgels in this study11. In this way, less material creates larger pores with a variety of different pore geometries while maintaining the stability of the 3D scaffold. The system consists of two types of microgel rods with complementary primary amine and epoxy functional groups that are consumed within the interlinking reaction when coming in contact with each other. The functional groups that do not participate in the interlinking process remain active and can be used for selective post-modification with cell adhesive peptides or other bioactive factors. Fibroblast cells attach, spread, and proliferate when cultured inside the 3D scaffolds, first growing on the microgel surface and filling most of the macropores after 5 days. A preliminary co-culture study of human fibroblasts and human umbilical vein endothelial cells (HUVECs) showed promising results for the formation of vessel-like structures within the interlinked 3D scaffolds11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABIL EM 90</td>
			<td>Evonik</td>
			<td>144243-53-8</td>
			<td>non-ionic surfactant</td>
		</tr>
		<tr>
			<td>2-Aminoethyl methacrylate hydrochloride</td>
			<td>TCI Chemicals</td>
			<td>A3413</td>
			<td>&#62;98.0%(T)(HPLC)</td>
		</tr>
		<tr>
			<td>8-Arm PEG-acrylate 20 kDa</td>
			<td>Biochempeg Scientific Inc.</td>
			<td>A88009-20K</td>
			<td>&#8805; 95 %</td>
		</tr>
		<tr>
			<td>AutoCAD 2019</td>
			<td>Autodesk</td>
			<td></td>
			<td>computer-aided design (CAD) software; modeling of microfluidic designs</td>
		</tr>
		<tr>
			<td>CHROMAFIL MV A-20/25 syringe filter</td>
			<td><s>CHROMAFIL</s>Carl Roth GmbH+Co.KG</td>
			<td>XH49.1</td>
			<td>pore size 0.20 &#181;m; Cellulose Mixed Esters (MV)</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Marienfeld-Superior</td>
			<td></td>
			<td>type No. 1</td>
		</tr>
		<tr>
			<td>EMS Swiss line core sampling tool 0.75 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td></td>
			<td>0.77 mm inner diameter, 1.07 mm outer diameter</td>
		</tr>
		<tr>
			<td>Ethanol absolut</td>
			<td>VWR Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FL3-U3-13Y3M 150 FPS series high-speed camera</td>
			<td>FLIR Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresceinamine isomer I</td>
			<td>Sigma-Aldrich</td>
			<td>201626</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Thermo Fisher Scientific</td>
			<td>46424</td>
			<td></td>
		</tr>
		<tr>
			<td>25G x 5/8&#8217;&#8217; 0,50 x 16 mm needles</td>
			<td>BD Microlance 3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycidyl methacrylate</td>
			<td>Sigma-Aldrich</td>
			<td>779342</td>
			<td>&#8805;97.0% (GC)</td>
		</tr>
		<tr>
			<td>GRGDS-PC</td>
			<td>CPC Scientific</td>
			<td>FIBN-015A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 1000 Series Gastight syringes</td>
			<td>Thermo Fisher Scientific</td>
			<td>10772361/10500052</td>
			<td>PFTE Luer-Lock</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>1,04,367</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate</td>
			<td>Sigma-Aldrich</td>
			<td>900889</td>
			<td>&#8805;95 %</td>
		</tr>
		<tr>
			<td>Motic AE2000 trinocular microscope</td>
			<td>Ted Pella, Inc.</td>
			<td>22443-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Novec 7100</td>
			<td>Sigma-Aldrich</td>
			<td>SHH0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>O9755</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>VWR Chemicals</td>
			<td>24679320</td>
			<td></td>
		</tr>
		<tr>
			<td>Pavone Nanoindenter Platform</td>
			<td>Optics11Life</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Tubing 0.38&#215;1.09mm medical grade</td>
			<td>dropletex</td>
			<td></td>
			<td>ID 0.38 mm OD 1.09 mm</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td>190764</td>
			<td>ACS reagent, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>Protein LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>30108132</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump 11 Pico Plus Elite Programmable Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>11530586</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 silicone elastomer kit</td>
			<td>Dow SYLGARD</td>
			<td>634165S</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro-(1H,1H,2H,2H-perfluoroctyl)-silane</td>
			<td>Sigma-Aldrich</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>UVC LED sterilizing box</td>
			<td>UVLED Optical Technology Co., Ltd.</td>
			<td>9S SZH8-S2</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64010/64010fig02.jpg" /> 
Figure 2: Macroporous crosslinked scaffold structure. (A) 3D projection of a 500 &#181;m confocal microscopy Z-stack of the interlinked macroporous scaffold. Scale bar represents 500 &#181;m. (B) Interlinked scaffold composed of ~10,000 microgel rods on a cover glass taken directly out of water. Scale bar repre...

Watch this Scientific Journal Video about Interlinked Macroporous 3D Scaffolds from Microgel Rods at JoVE.com

Interlinked Macroporous 3D Scaffolds from Microgel Rods

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 ...

interlinked-macroporous-3d-scaffolds-from-microgel-rods

Research

JoVE Journal

Bioengineering

2.0K Views.  DWI - Leibniz Institute for Interactive Materials. 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.

Video: Interlinked Macroporous 3D Scaffolds from Microgel Rods

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce &gt;85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with &gt;85% transduction.

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by ...