Name: controlling particle fraction in microporous annealed particle scaffolds for 3d cell culture
Uploaded: 2022-10-28T14:50:00
Description: Watch this Scientific Journal Video about Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture at JoVE.com

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

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 ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

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 ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

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 ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

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 ...

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 ...

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro

Particle image velocimetry (PIV) is used in a wide variety of fields, due to the opportunity it provides for precisely visualizing and quantifying flows across a large spatiotemporal range. However, its implementation typically requires the use of expensive and specialized instrumentation, which limits its broader utility. Moreover, within the field of bioengineering, in vitro flow visualization studies are also often further limited by the high cost of commercially sourced tissue phantoms that recapitulate desired anatomical structures, particularly for those that span the mesoscale regime (i.e., submillimeter to millimeter length scales). Herein, we present a simplified experimental protocol developed to address these limitations, the key elements of which include 1) a relatively low-cost method for fabricating mesoscale tissue phantoms using 3-D printing and silicone casting, and 2) an open-source image analysis and processing framework that reduces the demand upon the instrumentation for measuring mesoscale flows (i.e., velocities up to tens of millimeters/second). Collectively, this lowers the barrier to entry for nonexperts, by leveraging resources already at the disposal of many bioengineering researchers. We demonstratethe applicability of this protocol within the context of neurovascular flow characterization; however, it is expected to be relevant to a broader range of mesoscale applications in bioengineering and beyond.

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro

Here we present simplified methods for fabricating transparent neurovascular phantoms and characterizing the flow therein. We highlight several important parameters and demonstrate their relationship to field accuracy.

Particle image velocimetry (PIV) is used in a wide variety of fields, due to the opportunity it provides for precisely visualizing and quantifying flows ...

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 ...