The authors would like to thank Prof. Paul Van Tassel and Prof. Chinedum Osuji for their thoughtful conversations and materials science expertise. Funding for this work was provided by the Dubinsky New Initiative Award and National Institutes of Health NIBIB BRPR01 EB16629-01A1.

Please read Material Safety Data Sheet (MSDS) of all materials prior to use and use safety precautions at all times.
1. Synthesis of Zinc Oxide Needles
<ol>
	<li>Prepare 250 mL of a 0.04 M Zn(NO3)2*6H2O solution by adding 2.9749 g of zinc nitrate to 250 mL of water.</li>
	<li>Prepare 150 mL of 1 M NaOH by adding 6 g of NaOH to 150 mL of water.</li>
	<li>Set up a mineral oil bath on a hot plate with stirrer, and submerge a 500-mL round bottom flask into the oil bath at room temperature.</li>
	<li>Add 250 mL of Zn(NO3)2*6H2O to the flask and begin stirring the reaction.</li>
	<li>Add 150 mL NaOH solution (a white precipitate will form briefly and then disappear as the two solutions continue to mix). Stir for 2 h uncovered.</li>
	<li>Heat the flask to 55 - 60 &#176;C and continue stirring for 24 h.</li>
	<li>Turn off the heat and allow solution to cool to room temperature while stirring.</li>
	<li>Filter the solution on a B&#252;chner funnel with an 11 &#181;m pore size and allow to dry overnight, uncovered. When the filter paper is no longer wet from the poured solution and a white powder has formed over the funnel holes, the particles are fully dried.</li>
	<li>Collect ZnO needles and prepare for scanning electron microscopy (SEM) to confirm the needle-like morphology. Briefly, mount carbon tape onto a pin stub and use a metal spatula to smear the ZnO needles onto the carbon tape. Sputter coat with 8 mm iridium at a density of 22.4 g/cm3 and acquire images at 10 kV.</li>
</ol>
2. Addition of Sacrificial Zinc Layer on Microscope Slides for HCl Induced Release of PEG
<ol>
	<li>Prepare zinc acetate solution by dissolving 1.756 g of zinc acetate in 200 mL of methanol.</li>
	<li>Clean 3&#34; x 1&#34; plain microscope slides with 70% ethanol and a disposable wipe. Allow to air dry for 10 min.</li>
	<li>Place a 150-mm glass Petri dish onto a hot plate and preheat to 150 - 160 &#176;C.</li>
	<li>Using a glass pipet with a pipet bulb, hold the glass slides with tweezers and coat slides by applying 5 drops of zinc acetate, forming a thin layer dispersed on the slide. Allow excess solution to drip back into stock solution.</li>
	<li>Place the slide in the pre-heated Petri dish (150 - 160 &#176;C) with the zinc acetate coated side facing up. Leave on the hotplate for 15 min.</li>
	<li>Remove the slide with tweezers and allow it to cool to room temperature. The slide will appear to be coated with white streaks.</li>
	<li>Remove any excess zinc that is remaining on the slides using a disposable wipe to remove prominent white streaks. The surface should be uniform.</li>
	<li>Expose the the prepared slides to UV light in a biosafety hood for at least 1 h to ensure sterility. 
	Caution: UV light is harmful to eyes and exposed skin. Avoid direct exposure to eyes or skin and turn off electrical supply when not using.</li>
</ol>
3. Preparation of Silicone Isolators
<ol>
	<li>Cut silicone sheet into squares less than 1&#34; x 1&#34; (isolators must be able to fit in a 6-well dish).</li>
	<li>Punch 8 - 12 mm holes in the center of the squares with a biopsy punch.</li>
	<li>Autoclave silicone isolators for 20 min at 121.0 &#176;C and 1.12 kg/cm.</li>
</ol>
4. Preparation of PEG solution and PEG Gel Synthesis
Note: Functionalization and polymerization of PEG has been extensively explored and detailed previously by our lab and others.8,10,11,13,14,15,16,17
<ol>
	<li>Combine the RGD cell adhesive peptide with acryloyl-PEG-NHS in 50 mM sodium bicarbonate (pH 8.5) at a 1:1 molar ratio to enable functionalization of the amine terminus of the peptide with the acrylate moiety, a biochemical reaction that has been previously characterized to yield greater than 85% efficiency.17</li>
	<li>Prepare photo-initiator by mixing 2,2-Dimethoxy-2-phenylacetophenone with 1-Vinyl-2-pyrrolidone at a concentration of 300 mg/mL.</li>
	<li>In separate 1.5 mL tubes, weigh out the following: 20 mg of ZnO needles, 12.5 mg of PEG-DA, and 2.5 mg of PEG-RGD.</li>
	<li>Suspend 20 mg of ZnO needles in 270 &#181;L of 10% FBS/PBS solution; vortex to mix (approximately 15 s).</li>
	<li>Briefly (&#60;1 s) spin the solution in a benchtop mini centrifuge to bring any ZnO aggregates to the base of the tube.</li>
	<li>Collect 250 &#181;L from the top of the ZnO solution, to ensure that aggregates remain at the bottom of the tube. Combine with PEG-DA and PEG-RGD to create a PEG solution with approximately 2.5 mM RGD. Vortex to mix (approximately 15 s).</li>
	<li>Add 2 &#181;L of acetophenone/n-vinyl pyrrolidone photo-initiator and vortex briefly to mix (approximately 5 s).</li>
	<li>Add 20 &#181;L of the polymer solution along the center of the ZnO coated slides.</li>
	<li>Slowly lower a second slide on top, with the ZnO coating face down (the PEG solution should be between two ZnO coated layers). Try to prevent the formation of any air bubbles. Move the slides laterally along the major axis to allow any bubbles to escape and to spread the solution to a thin layer. Cover the majority of the slide with the polymer solution. Create a slight overhang with the top slide to ensure that the slides can be pulled apart in later steps.</li>
	<li>Crosslink the slides under a 365 nm UV lamp (approximately 10 mW/cm2) for 15 min.</li>
</ol>
5. Release of PEG Gels from Glass Slides
<ol>
	<li>Apply pressure to the overhanging slide and manually pull the two slides apart at a slow pace in order avoid cracking the slides. Allow the gels to dry for at least 5 min, or overnight.</li>
	<li>Place the slide in a sterile 25 mm glass Petri dish and gently pour the 1 M HCl solution onto the slide, using only enough to cover the slide (approximately 10 - 20 mL). Gently rock the Petri dish; the gel should begin to lift off the slide as the HCl dissolves the sacrificial zinc coating and needles. Once the gel is free from the slide, pour the HCl back into the stock solution.</li>
	<li>Rinse the gel by gently pouring approximately 25 mL of 1x PBS into the Petri dish until the slide and gel are submerged. Carefully pour off PBS into a waste container.</li>
	<li>Gently pour approximately 25 mL of 1x PBS into the Petri dish until the gel is floating in the solution.</li>
	<li>Using tweezers, carefully slide the silicone isolator under the gel and lift the gel onto it.</li>
	<li>Transfer the isolator and the gel into a 6-well dish filled with sterile 1x PBS.</li>
	<li>Repeat this process until all of the gels have been removed from the slides and are soaking in PBS. Expose the prepared gels to UV light in a biosafety hood for at least 1 h to ensure sterility.</li>
</ol>
6. Seeding Cell Bilayers
<ol>
	<li>Prepare A549 cells for seeding. Briefly, rinse a 75 cm2 flask with 5 mL of 1x PBS, add 3 mL of 0.25% Trypsin-EDTA and let sit for 2 - 3 min at 37 &#176;C.
	<ol>
		<li>Mechanically agitate the flask, quench the reaction with 3 mL Dulbecco&#39;s Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin-streptomycin, and collect into a 15-mL conical flask. Rinse with an additional 4 mL of complete Dulbecco&#39;s Modified Eagle Media and collect into the same conical. Spin cells at 475 x g for 6 min.</li>
	</ol>
	</li>
	<li>While cells are spinning down, add a small drop of complete Dulbecco&#39;s Modified Eagle Medium to each well of a 6-well plate. Use tweezers to transfer the silicone isolators with the gels into the new 6-well plate, such that the gel is resting on the drop of media. Now that gels are spread into a thin layer, check to ensure that there are no large holes visible to the eye. This should be done immediately prior to cell seeding to avoid drying and cracking of the gels.</li>
	<li>Resuspend cells in Dulbecco&#39;s Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin-streptomycin at a concentration of 6 x 105 cells/mL. Add the cell suspension drop wise to the center of the gel, trying to maintain a meniscus in the punched out area of the silicone membrane. Allow cells to adhere for 4 h.</li>
	<li>After 4 h, add 0.5 mL of Dulbecco&#39;s Modified Eagle Complete Medium to the well and incubate overnight at 37 &#176;C to allow for complete adhesion.</li>
	<li>The following day, prepare HUVECs for cell seeding.
	<ol>
		<li>Rinse a 75 cm2 flask with 5 mL of 1X PBS, add 3 mL of 0.25% Trypsin-EDTA and let sit for 2 - 3 min at 37 &#176;C.</li>
		<li>Mechanically agitate the flask, quench the reaction with 3 mL M199 media with 20% fetal bovine serum, 1% penicillin-streptomycin, and 1% growth supplement, and collect into a 15-mL conical.</li>
		<li>Rinse with 4 mL of M199 complete media and collect into the same conical. Spin cells at 475 x g for 6 min.</li>
	</ol>
	</li>
	<li>While cells are spinning down, add a small drop of complete medium 199 to each well in a new 6-well plate. Place a new silicone isolator in the well with the drop of media in the center of the silicone.</li>
	<li>Using tweezers, carefully flip the silicone isolator supporting the PEG gel with A549 cells onto the isolator in the new 6-well plate.</li>
	<li>Resuspend HUVECs at a concentration of 6 x 105 cells/mL and add the cell suspension to the center of the flipped gel drop wise, trying to maintain a meniscus on the gel within the punched out area of the silicone membrane. Allow cells to adhere for 2 h.</li>
	<li>After 2 h, gently add 2 mL of M199 complete media to each well.</li>
</ol>
7. Immunofluorescence
<ol>
	<li>Carefully remove media from gels with a manual pipette and add approximately 500 &#181;L of 4% paraformaldehyde (PFA) to the center of the gel where the cells are seeded. Let sit for 30 min at room temperature. 
	&#8203;Caution: PFA is a toxic chemical; wear protection and ensure handling is done in a biological safety cabinet.</li>
	<li>Carefully remove the PFA and add approximately 500 &#181;L of 2% BSA in PBS to each gel. Let sit for 1 h at room temperature.</li>
	<li>Carefully remove BSA. Prepare primary antibodies at a dilution of 1:100 in 2% BSA in PBS, and add 500 &#181;L to each gel. Let sit for 1 h at room temperature. Rinse gently with 500 &#181;L of 1x PBS.
	<ol>
		<li>Repeat the same process with the secondary antibodies. Use following antibodies: 1) anti-human CD144 (VE-Cadherin) clone 16B1; 2) anti-human CD324 (E-Cadherin) clone 6714; 3) anti-human CD31 (PECAM-1) clone C-20; 4) anti-A549; 5) anti-mouse FITC; 6) anti-goat Alexa Fluor 647. To visualize F-actin, add 500 &#181;L of Phalloidin (10 &#181;g/mL in PBS) for 20 min.</li>
	</ol>
	</li>
	<li>Carefully remove secondary antibodies or phalloidin and add 500 &#181;L of DAPI (0.1 &#181;g/mL) for 20 min. Carefully remove DAPI solution and gently add 1.5 mL of 1x PBS to each well so that gels are in suspension.
	<ol>
		<li>Using tweezers and silicone isolators, transfer one gel to a glass slide. Do this for each gel immediately before imaging, and replace gels into PBS solution after imaging.</li>
		<li>Alternatively, mount gels using a DAPI mounting medium. Seal well the samples with nail polish, acquire images within 2 days to prevent drying and cracking of the gel. Refer to Table 1 for troubleshooting.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The protocol detailed here has allowed us to create a tunable PEG hydrogel to serve as a biomimetic BM scaffold. Specifically, by varying PEG molecular weights, peptide conjugation strategies, and zinc oxide microcrystalline structures or concentrations, the elastic modulus, biochemical properties, and porous structure of the hydrogels can be modified, respectively. The ultrathin PEG scaffold features a higher pore density and a smaller pore diameter that is more mimetic of the features found in in vivo basement...

Extracellular matrices (ECM) make up the protein scaffolds that support cell attachment and serve as barriers between distinct cell types and are an essential component of complex tissues and organs. In contrast to interstitial connective tissue, the basement membrane (BM) is a specialized type of ECM that acts as a barrier to divide tissue compartments from one another. BMs are approximately 100 &#181;m thick, and therefore allow for direct and indirect communication between cells on either side. Two common examples of BMs are vascular BMs, found in the microvascular wall between pericytes and endothelial cells, and airway BMs that are found between endothelial and epithelial cells. BMs serve an important role in regulating cell function, such as cell polarity and migration, in health and disease.1 The composition, stiffness, architecture, and porosity of BMs varies across organ systems to facilitate distinct physiological functions. For example, BM pores are critical for maintaining cell-cell communication, soluble molecule diffusion, and for migration of immune cells during inflammation or bacteria during infection. In the airways, pores span the full thickness of the BM, with diameters ranging from 0.75 to 3.86 &#181;m.2
The thin nature of the BM ensures that although cell types are physically separated from one another, intercellular communication via paracrine- and contact-mediated signaling is preserved. Thus, to study human disease in vitro, researchers have relied on porous permeable support inserts to culture cellular bilayers.3 These models have been critical for understanding the cellular communication that plays a role in health and disease.3,4,5,6,7 Permeable support inserts satisfy the basic requirements for understanding how cell-cell signaling regulates physiological processes, such as leukocyte recruitment and bacterial infiltration; however, the inserts have significant limitations and fail to mimic a human BM. Permeable support inserts lack both mechanical and biochemical tunability, and the simplistic porous structure does not mimic the fibrous structure that creates the irregular pores typical of BMs. Therefore, there is a growing need for tunable systems that can recreate the native BM properties that influence cellular processes.
Polymer-based substrates are ideal candidates for the development of biomimetic BMs to study cellular bilayers in a context that more closely mimics the in vivo environment.8,9,10,11,12 Polymers are mechanically tunable and can be chemically modified to incorporate biomimetic peptide fragments.11,12,13 The bioinert polymer polyethylene glycol (PEG) can be used to construct biomimetic BMs, and recent work has detailed the synthesis of mechanically tunable PEG arginine-glycine-aspartic acid (RGD) gels with porous networks that support cell growth and inflammatory cell chemotaxis.14 Although published PEG-based substrates provided a more realistic model of a human ECM than permeable supports, many of these models are extremely thick, with a depth of roughly 775 &#181;m that limits the ability to create bilayer cultures with cell-cell contacts.14
Here, we present a protocol for the creation of a PEG polymer-based BM mimic that overcomes many of the limitations of current cell bilayer culture technologies. We have developed a templating method that incorporates zinc oxide, an extensively used material for the manufacture of microcrystalline production, into the polymer during synthesis and crosslinking, which is subsequently and selectively removed from the resulting bulk polymer. This process generates a random porous network, mimicking the tortuous and interconnected pore network of human BMs. Further, the porosity can be altered by changing the size and shape of the zinc oxide microcrystals via modification of the reaction stoichiometry during needle production. The technique developed here creates an ultrathin hydrogel that mimics the thickness of human BM. Lastly, the mechanics, the porosity, and the biochemical composition of these BM-like constructs can easily be altered to generate a microenvironment that is most similar to that seen in vivo.

<table><tbody><tr><td>1M Hydrogel Chloride (HCl)</td><td>EMD</td><td>HX0603-75 2.5L</td><td>Sterile. Use in fume hood with eye protection and gloves.</td></tr><tr><td>1X PBS</td><td>Gibco</td><td>14040-133 500 mL</td><td>None</td></tr><tr><td>Zinc Nitrate Hexahydrate (Zn(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>228737-500g</td><td>Use with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Macron Chemicals</td><td>278408-500g</td><td>Use with eye protection and gloves.</td></tr><tr><td>Zinc Acetate Dihydrate ((CH&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;Zn&lt;sup&gt;2+&lt;/sup&gt;&amp;bull;2H2O)</td><td>Fisher Scientific</td><td>AC45180010 1 kg</td><td>Use with eye protection and gloves.</td></tr><tr><td>Methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>J.T. Baker</td><td>9070-05 4L</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>VWR Life Science Seradigm Premium Grade FBS</td><td>VWR</td><td>97068-085</td><td>Sterile filter. 5 mL FBS in 45 mL PBS</td></tr><tr><td>Mineral oil</td><td>CVS&amp;nbsp;</td><td>PLD-B280B</td><td>None</td></tr><tr><td>Round bottom flask</td><td>ChemGlass</td><td></td><td>N/A</td></tr><tr><td>Thermometer</td><td></td><td></td><td>N/A</td></tr><tr><td>Stir bar</td><td></td><td></td><td>N/A</td></tr><tr><td>Plain precleaned microscope slides 3&quot;x1&quot;x1&quot; mm thick</td><td>Thermo Scientific</td><td>420-004T</td><td>Spray with ethanol and let dry prior to use.</td></tr><tr><td>Glass pasteur pipets</td><td></td><td></td><td>N/A</td></tr><tr><td>1 mL rubber bulbs</td><td></td><td></td><td>N/A</td></tr><tr><td>Plastic 100 mm petri dishes</td><td></td><td></td><td>N/A</td></tr><tr><td>Sterile forceps</td><td></td><td></td><td>N/A</td></tr><tr><td>Silicone isolators</td><td></td><td></td><td>0.8 mm thick</td></tr><tr><td>Polydimethylsiloxane (PDMS) punches</td><td></td><td></td><td>N/A</td></tr><tr><td>Glass bottles</td><td></td><td></td><td>N/A</td></tr><tr><td>6 well plates</td><td>Cellstar</td><td>657 160</td><td>N/A</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Stirrer-hot plate</td><td>VWR Dya-Dual</td><td>12620-970</td><td>Use with eye protection and gloves.</td></tr><tr><td>2,2-Dimethoxy-2-phenylacetophenone (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;COC(OCH&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>24650-42-8</td><td>Use with eye protection and gloves.</td></tr><tr><td>1-Vinyl-2-pyrrolidone (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;9&lt;/sub&gt;NO)</td><td>Aldrich</td><td></td><td>Use with eye protection and gloves.</td></tr><tr><td>Polyethylene Glycol 10,000 (H(OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;10,000&lt;/sub&gt;OH)</td><td>Fluka</td><td>81280-1kg</td><td>Use with eye protection and gloves.</td></tr><tr><td>RGDS</td><td>Life Tein</td><td>180190</td><td>Use with eye protection and gloves.</td></tr><tr><td>Blak-Ray long wave UV lamp</td><td>UVP</td><td>Model B 100AP</td><td>N/A</td></tr><tr><td>Eppendorf tubes</td><td>USA Scientific</td><td>1615-5500</td><td>N/A</td></tr></tbody></table>

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.

PEG-RGD hydrogels were formed by sandwiching the polymer solution between two sacrificial zinc oxide layers and creating pore templates with zinc oxide needles. Sacrificial zinc oxide components were then removed with hydrochloric acid, generating ultrathin PEG hydrogels with continuous pores (Figure 1). The morphology of zinc oxide needles was confirmed by scanning electron microscopy (SEM), and the average length and width were determined to be 3.92 &#177;&...

Watch this Scientific Journal Video about Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture at JoVE.com

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

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 ...

ultrathin-porated-elastic-hydrogels-as-biomimetic-basement-membrane

Research

JoVE Journal

Bioengineering

7.7K Views.  Yale University. Actuele modellen van de cultuur van de dubbelgelaagde toegestaan niet voor functionele in vitro studies die in vivo microenvironments na te bootsen. Met behulp van polyethyleenglycol en een methode van zinkoxide templating, beschrijft dit protocol de ontwikkeling van een uiterst dunne biomimetische kelder membraan met afstembare stijfheid, porositeit en biochemische samenstelling die nauw samen van de Nabootsers van in vivo extracellulaire matri...

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

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 ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

3D Bioprinting for Rapid Vascular Network Fabrication Using Vessels-on-a-Plate

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

Formation and Attachment of FN-Silk Membranes to Inserts of Cell Culture 

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Author Spotlight: Enhancing In Vitro Cell Culture Models with Recombinant Functionalized Spider Silk Membranes

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

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

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