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.

<ol>
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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

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...

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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 ...

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

Research

JoVE Journal

Bioengineering

2.3K Views.  Duke University. Korrelige materialen, zoals MAP-steigers, zijn poreuze injecteerbare materialen gemaakt van onderling verbonden hydrogeldeeltjes van microformaat. Het beheersen van de deeltjesfractie is van cruciaal belang voor materiaaleigenschappen en cellulaire reacties. Variabele deeltjesfracties in MAP-scaffolds resulteren in een reeks scaffold-eigenschappen en celresponsen, maar deze techniek stelt gebruikers in staat om de uiteindelijke deeltjesfractie te definiëren in de MAP-scaffolds die ze maken.

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

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

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

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.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

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 ...

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

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

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.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

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

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

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

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.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

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.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

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.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of ...

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.

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 ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e.,&#160;annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

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

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that ...

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

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