This work was supported by the National Science Foundation through the UPenn MRSEC program (DMR-1720530) and graduate research fellowships (to V.G.M and M.E.P.) and the National Institutes of Health (R01AR077362 to J.A.B.).

The authors have no competing financial interests.

Herein, methods to fabricate granular hydrogels using extrusion fragmented microgels and packing by either centrifugation or vacuum-driven filtration are described. Compared to other microgel fabrication methods (i.e., microfluidics, batch emulsions, electrospraying, photolithography), extrusion fragmentation microgel fabrication is highly rapid, low-cost, easily scalable, and amenable to a wide variety of hydrogel systems. Further, this protocol is highly repeatable with minimal batch-to-batch variability, which was cha...

Granular hydrogels are fabricated through the packing of hydrogel particles (i.e., microgels) and are an exciting class of biomaterials with many advantageous properties for biomedical applications1,2,3. Due to their particulate structure, granular hydrogels are shear-thinning and self-healing, allowing for their use as extrusion printing (bio)inks, granular supports for embedded printing, and injectable therapeutics4,5,6,7,8,9. Additionally, the void space between microgels provides a microscale porosity for cell movement and molecular diffusion8,10,11. Further, multiple microgel populations can be combined into a single formulation to allow for enhanced tunability and material functionality8,10,12,13. These important properties have motivated the rapid expansion of granular hydrogel development in recent years.
There is a range of methods available to form microgels towards granular hydrogel fabrication, each with its own advantages and disadvantages. For example, microgels are often formed from water-in-oil emulsions using droplet microfluidics4,11,13,14,15,16,17, batch emulsions7,18,19,20,21,22, or electrospraying6,23,24,25. These methods yield spherical microgels with either uniform (microfluidics) or polydisperse (batch emulsions, electrospraying) diameters. There are some limitations to these water-in-oil emulsion fabrication methods, including potentially low-throughput production, the need for low-viscosity hydrogel precursor solutions, and the high cost and resources for setup. Additionally, these protocols may require harsh oils and surfactants that must be washed from the microgels using procedures that add processing steps, and may be difficult to translate to sterile conditions for biomedical applications in many labs. Removing the need for water-in-oil emulsions, (photo)lithography can also be used, where molds or photomasks are used to control the curing of microgels from hydrogel precursor solutions1,26,27. Like microfluidics, these methods may be limited in their production throughput, which is a major challenge when large volumes are needed.
As an alternative to these methods, mechanical fragmentation of bulk hydrogels has been used to fabricate microgels with irregular sizes19,28,29,30,31,32. For example, bulk hydrogels can be pre-formed and subsequently passed through meshes or sieves to form fragmented microgels, a process which has even been done in the presence of cells within microgel strands33,34. Bulk hydrogels have also been processed into microgels with mechanical disruption using techniques such as grinding with mortar and pestle or through the use of commercial blenders35,36,37. Others have also used mechanical agitation during hydrogel formation to fabricate fragmented microgels (i.e., fluid gels)31.
The methods herein expand on these mechanical fragmentation techniques and present a simple approach to fabricate microgels with extrusion fragmentation, using photocrosslinkable hyaluronic acid (HA) hydrogels as an example. Extrusion fragmentation uses only syringes and needles to fabricate fragmented microgels in a low-cost, high throughput, and easily scalable method that is appropriate for a wide range of hydrogels19,32. Further, methods to assemble these fragmented microgels into granular hydrogels are described using either centrifugation (low packing) or vacuum-driven filtration (high packing). Lastly, the application of these fragmented granular hydrogels is discussed for use as an extrusion printing ink. The goal of this protocol is to introduce simple methods that are adaptable to a wide variety of hydrogels and can be implemented in virtually any laboratory interested in granular hydrogels.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Plastic Conical Centrifuge Tube</td>
			<td>Corning</td>
			<td>430766</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G NT Premium Series Dispensing Tip</td>
			<td>Jensen Global</td>
			<td>JG30-0.5HPX</td>
			<td>Catalog Number listed here is for 30 G, 0.5&#34; needle. Various sizes are available.</td>
		</tr>
		<tr>
			<td>BD Disposable Syringes with Luer-Lok Tips (3 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-823-435</td>
			<td>Catalog Number listed here is for 3 mL syringe. Various sizes are available (14-823-XXX).</td>
		</tr>
		<tr>
			<td>Black folders</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (18 G, 0.5&#34;)</td>
			<td>Grainger</td>
			<td>5FVH5</td>
			<td>Catalog Number listed here is for 18 G, 0.5&#34; needle. Various sizes are available.</td>
		</tr>
		<tr>
			<td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (23 G, 0.5&#34;)</td>
			<td>Grainger</td>
			<td>5FVJ3</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (27 G, 1.5&#34;)</td>
			<td>Grainger</td>
			<td>5FVL0</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>14190-250</td>
			<td>Catalog Number listed here is for a case of 10 x 500 mL bottles.</td>
		</tr>
		<tr>
			<td>Durapore Membrane Filter, 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>GVWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent or confocal microscope</td>
			<td>Various Vendors</td>
			<td></td>
			<td>To visualize microgels and granular hydrogels</td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Safe-Lock Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Extrusion printer</td>
			<td>Custom-built</td>
			<td></td>
			<td>Other extrusion printers can be use,d such as commercially available BIOX.</td>
		</tr>
		<tr>
			<td>Filter Adapters</td>
			<td>Fisher Scientific</td>
			<td>05-888-107</td>
			<td>Catalog Number listed here is for a set of multiple sizes. Various sizes are available (05-888-XXX).</td>
		</tr>
		<tr>
			<td>Filter Flask</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran (2 MDa)</td>
			<td>Sigma-Aldrich</td>
			<td>52471</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>&#34;Analyze Particles&#34; information link: https://imagej.nih.gov/ij/docs/menus/analyze.html</td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lock Tip Caps</td>
			<td>Integrated Dispensin g Solutions</td>
			<td>9991329</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal spatula for scooping</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Various Vendors</td>
			<td></td>
			<td>Capable of speed up to 18,000 x g</td>
		</tr>
		<tr>
			<td>Microscoft Execl</td>
			<td>Microsoft</td>
			<td></td>
			<td>Other programs can be used, such as Google Slides.</td>
		</tr>
		<tr>
			<td>OmniCure S2000 Spot UV Curing System</td>
			<td>Excelitas Technologies</td>
			<td>S2000</td>
			<td>Different light systems may be used to fabricate bulk hydrogels if desired.</td>
		</tr>
		<tr>
			<td>Porcelain Buchner Funnel with Fixed Perforated Plate</td>
			<td>Fisher Scientific</td>
			<td>FB966C</td>
			<td>Catalog Number listed here is for 56mm diameter plate. Various sizes are available.</td>
		</tr>
		<tr>
			<td>Radiometer</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repetier Host</td>
			<td>Hot-World GmbH &#38; Co. KG</td>
			<td></td>
			<td>3D printing software</td>
		</tr>
		<tr>
			<td>Screw-based extrusion printer</td>
			<td>Various Vendors</td>
			<td></td>
			<td>This study used a custom-modified 3D FDM printer (Velleman K8200). Many alternatives are available.</td>
		</tr>
		<tr>
			<td>Solidworks/CAD software</td>
			<td>Dassault Syst&#232;mes SolidWorks Corporation</td>
			<td></td>
			<td>Other programs can be used, such as Blender or TinkerCAD.</td>
		</tr>
		<tr>
			<td>Tubing to Connect Filter Flask to Vacuum Line</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV Eye Protection (i.e., safety glasses)</td>
			<td>Various Vendors</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fragmenting bulk hydrogels and processing into granular hydrogels for biomedical applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

Representative results from these protocols are shown in Figure 3 and Figure 6. Extrusion fragmentation yields microgels with jagged, polygon shapes with diameters ranging from 10-300 &#181;m (Figure 3). Further, circularity ranges from 0.2 (not circular) to almost 1 (perfect circle), and the aspect ratio ranges from 1-3 (Figure 3). These parameters describe the irregular and jagged microgel shapes form...

Watch this Scientific Journal Video about Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications at JoVE.com

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

fragmenting-bulk-hydrogels-processing-into-granular-hydrogels-for

Research

JoVE Journal

Bioengineering

5.3K Views.  University of Pennsylvania. This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications. 

Video: Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...