Name: Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture (Video) | JoVE Journal
Uploaded: 2022-10-28T14:50:00.000+00:00
Duration: 8 min 11 s
Description: Learn to control particle fractions in MAP scaffolds for reproducible 3D cell culture. This method enables precise tuning of material properties and cellular responses in tissue engineering applications.

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5 minutes

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.

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<li>Muir, V. G., Qazi, T. H., Shan, J., Groll, J., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+microgel+fabrication+technique+on+granular+hydrogel+properties%5BTitle%5D)+AND+%22ACS+Biomaterials+Science+%26+Engineering%22%5BJournal%5D)">Influence of microgel fabrication technique on granular hydrogel properties.</a> ACS Biomaterials Science & Engineering. 7 (9), 4269-4281 (2021).</li>
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<li>Anderson, A. R., Nicklow, E., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle+fraction+as+a+bioactive+cue+in+granular+scaffolds%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">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/pubmed?term=((Spatially+heterogeneous+epidermal+growth+factor+release+from+microporous+annealed+particle+%28MAP%29+hydrogel+for+improved+wound+closure%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry+B%22%5BJournal%5D)">Spatially heterogeneous epidermal growth factor release from microporous annealed particle (MAP) hydrogel for improved wound closure.</a> Journal of Materials Chemistry B. 9 (35), 7132-7139 (2021).</li>
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<li>Roosa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+synthesis+of+microgel+building+blocks+for+microporous+annealed+particle+scaffold%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Interfacial+bioorthogonal+crosslinking%5BTitle%5D)+AND+%22ACS+Macro+Letters%22%5BJournal%5D)">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/pubmed?term=((Cryogel+micromechanics+unraveled+by+atomic+force+microscopy-based+nanoindentation%5BTitle%5D)+AND+%22Advanced+Healthcare+Materials%22%5BJournal%5D)">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., Bergenståhl, B., Mattiasson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macroporous+elastic+polyacrylamide+gels+prepared+at+subzero+temperatures%3A+control+of+porous+structure%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry%22%5BJournal%5D)">Macroporous elastic polyacrylamide gels prepared at subzero temperatures: control of porous structure.</a> Journal of Materials Chemistry. 16 (41), 4065-4073 (2006).</li>
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<li>Isaac, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microporous+bio-orthogonally+annealed+particle+hydrogels+for+tissue+engineering+and+regenerative+medicine%5BTitle%5D)+AND+%22ACS+Biomaterials+Science+%26+Engineering%22%5BJournal%5D)">Microporous bio-orthogonally annealed particle hydrogels for tissue engineering and regenerative medicine.</a> ACS Biomaterials Science & Engineering. 5 (12), 6395-6404 (2019).</li>
<li>Truong, N. F., Lesher-Pérez, S. C., Kurt, E., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathways+governing+polyethylenimine+polyplex+transfection+in+Microporous+Annealed+Particle+scaffolds%5BTitle%5D)+AND+%22Bioconjugate+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Enhanced+in+vivo+delivery+of+stem+cells+using+microporous+annealed+particle+scaffolds%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">Enhanced in vivo delivery of stem cells using microporous annealed particle scaffolds.</a> Small. 15 (39), 1903147(2019).</li>
<li>Li, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cartilage+tissue+formation+through+assembly+of+microgels+containing+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells.</a> Acta Biomaterialia. 77, 48-62 (2018).</li>
</ol>

ARA and TS have filed a provisional patent on this technology.

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><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&quot;, Non-Sterile, Capped</td><td>SAI Infusion Technologies</td><td>B23-50</td><td></td></tr><tr><td>Bottle-top vacuum filter, 0.22 &amp;mu;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 &amp;mu;L max. volume</td><td>Rainin</td><td>17008609</td><td></td></tr><tr><td>Capillary-piston assemblies for positive-displacement pipettes, 25 &amp;mu;L max. volume</td><td>Rainin</td><td>17008605</td><td></td></tr><tr><td>Capillary-piston assemblies for positive-displacement pipettes, 250 &amp;mu;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)&amp;nbsp;</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&#039;s Modified Eagle&#039;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 &amp;lt;100 CFU/g, bacterial endotoxins &amp;lt;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 &amp;mu;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&amp;nbsp;</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&#039;s</td><td>XZ3601W</td><td></td></tr></tbody></table>

controlling particle fraction in microporous annealed particle scaffolds for 3d cell culture

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...

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 ...

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

Research

JoVE Journal

Bioengineering

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

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 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.

Self-reporting Scaffolds for 3-Dimensional Cell Culture

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

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.

Interlinked Macroporous 3D Scaffolds from Microgel Rods

This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications.

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

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.

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Micro-particle image velocimetry (μPIV) 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 for Velocity Profile Measurements of Micro Blood Flows

Learn to control particle fractions in MAP scaffolds for reproducible 3D cell culture. This method enables precise tuning of material properties and cellular responses in tissue engineering applications.

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

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