This work was supported by the Delaware COBRE programs in Drug Discovery and in Advanced Biomaterials funded by Institutional Development Awards from the National Institute of Generals Medical Sciences at the National Institutes of Health (P20GM104316 and P30 GM110758-01, respectively), the Pew Charitable Trusts (00026178), a National Science Foundation Career Award (DMR-1253906), the Burroughs Wellcome Fund (1006787), and the National Science Foundation IGERT SBE2 program at the University of Delaware (fellowship to L. Sawicki). The authors thank the Delaware Biotechnology Institute BioImaging Center at the University of Delaware for training and access to confocal microscopy, Ms. Katherine Wiley for assistance during the video shoot, Mr. Matthew Rehmann for generously providing hMSCs isolated from bone marrow, Prof. Christopher J. Kloxin and Mr. Stephen Ma for generously providing photomasks, and Prof. Wilfred Chen for the use of the automated plate reader.

<ol>
	<li>Kolb, H. C., Finn, M. G., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry:+Diverse+Chemical+Function+from+a+Few+Good+Reactions.">Click Chemistry: Diverse Chemical Function from a Few Good Reactions.</a> Angew Chemie - Int Ed. 40 (11), 2004-2021 (2001).</li><li>Xi, W., Scott, T. F., Kloxin, C. J., Bowman, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+in+Materials+Science.">Click Chemistry in Materials Science.</a> Adv Funct Mater. 24 (18), 2572-2590 (2014).</li><li>Azagarsamy, M. A., Anseth, K. 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The authors have nothing to disclose.

The procedure presented here demonstrates techniques to photoencapsulate cells within hydrogels formed by thiol&#8722;ene click chemistry and subsequently photopattern the gels with biochemical cues. The use of light to initially form hydrogels allows homogeneous mixing and suspension of the cells within the polymer solution prior to polymerization. Rapid polymerization &#39;locks&#39; the gel in the shape of the defined mold and encapsulates suspended cells within the hydrogel network. Gels also may be molded into numerous different shapes (e.g., glass slides or syringe tips) depending on the final application desired. For example, cells encapsulated for 3D culture in hydrogels attached to glass slides are particularly useful for imaging applications as light attenuation is limited within a thin sample. Syringe molds can be used for rapid encapsulation of cells, allowing a larger number of samples to be prepared in a short time (compared to glass slides) that can be used for experiments requiring large numbers of cells such as flow cytometry or qPCR. Subsequently, these gels then may be patterned with biochemical cues to elicit desired cellular responses such as differentiation or invasion.30,31
Assays for viability and metabolic activity indicate the survival of cells for the material system and patterning conditions presented. Note that metabolic activity was monitored until day 3 in non-degradable gels (RGKGRK peptide crosslink) to assess the initial effects of polymerization and patterning conditions on cell function. Additionally, a membrane integrity assay (live/dead viability/cytotoxicity staining) of hMSCs at 1 and 6 days post-encapsulation in gels formulated with a degradable peptide crosslink (GPQGIWGQ) supports that cells remain viable and spread at 1 week in culture. The viability of additional cell lines has been reported for photopatterning conditions32 similar to those used here and can be evaluated for the described hydrogel system using live/dead staining and metabolic activity assays. While we have not observed issues with cell viability using this materials system and related procedures (4.2 and 4.3), some cell types may be sensitive to free-radical and/or light exposure. In this case, users may consider using non-photoinitiated materials systems, such as azide-alkyne,12 FXIII,31 or Diels Alder-based hydrogel formation chemistries.33
Facile techniques to detect and quantify patterning of biochemical cues within gels also are presented (3.1 and 3.2). Ellman&#39;s assay is of particular interest because the reagents are commercially available and no extra synthetic processing steps or more expensive reagents (e.g., fluorescently-labeled peptide) are required. Ellman&#39;s assay can be used to precisely determine the modification of free thiols with biochemical cues under different photopatterning conditions, as well as to rapidly visualize patterns. For quantifying peptide incorporation, the thiol functional group concentration before and after patterning, as a quantitative measure of peptide incorporation, is directly assessed with Ellman&#39;s assay. While this type of quantification can be done with fluorescently-labeled peptides,34 imaging-based quantification requires more time-consuming handling and analysis steps (e.g., synthesis of a fluorescently-labeled peptide and generation of a calibration curve to relate fluorescence to peptide concentration using image analysis). For imaging peptide incorporation, Ellman&#39;s reagent can be directly applied to samples and immediately visualized. While limited to the x- and y-planes for pattern visualization, the technique can be used as a simple, routine method to determine if matrices containing free thiol groups have been patterned. It is important to note that Ellman&#39;s assay is not considered cytocompatible, so while it may be used to observe and quantify photopatterns, it cannot be done in the presence of cells. For imaging patterning in three dimensions and in the presence of cells, the conjugation of fluorescent peptides within hydrogel matrices remains a powerful and widely-used approach. Resolution of patterns can be evaluated in the x-, y-, and z-planes using confocal microscopy, and this method is cytocompatible so that cells within patterned regions or non-patterned regions can be identified. Taken together, Ellman&#39;s assay and imaging-based techniques are complementary tools for researchers to assess both quantitatively and qualitatively the photopatterning of biochemical cues within the materials system.
Photoclick, or more broadly photoinitiated, chemistries for the formation and modification of hydrogels in the presence of cells are numerous. The molding, encapsulation, and patterning techniques presented here are not limited to the described material system and may be applied to alternative light-based chemistries, such as thiol-alkyne,35 azide-alkyne,4 and other thiol&#8722;ene chemistries (e.g., thiol-norbornene),10,13 as well as with different photoinitiators, such as Irgacure 2959, Eosin Y, and camphorquinone. Note, users may need to adjust procedure parameters (e.g., incubation times, polymerization times, cell density) to ensure that conditions remain cytocompatible for these other systems. Since the patterning process requires diffusion of the alloc-modified peptide(s) into the hydrogel (2.2.3-2.2.5), this process may prove most useful for the addition of integrin-binding moieties (e.g., peptides or extracellular matrix protein fragments) to the hydrogel, where attachment of the ligand to the network is required for the generation of traction forces by the cell and full integrin activation.36 Note, for biomolecules that may be similarly active in solution or upon immobilization (e.g., growth factors or cytokines), the incubation step for moiety diffusion (~1 hr) into the hydrogel could lead to signaling events that convolute patterning results. Other methods have been established for growth factor immobilization or local sequestration for their patterning.37-39Additionally, pattern resolution is dictated by the control over light exposure. Here, photomasks allow creation of patterns through the gel depth and in the x- and y-planes; however, greater spatial control over patterning of biochemical cues within gels can be achieved with alternative methods of irradiation such as the use of a two-photon confocal microscope to generate patterns in the x-, y-, and z- planes.34,40 Finally, it is important to note that while the material system utilized within this procedure is only initially modified with biochemical cues, orthogonal photoclick chemistries could be used to allow alterations in matrix properties over time.12 The procedure and techniques presented here add diversity to the current approaches for creating synthetic matrices with well-defined and spatiotemporally-controlled properties. In particular, the commercial availability of reagents and materials used within this procedure will be useful to a wide range of researchers interested in the use of hydrogel-based biomaterials for applications in controlled cell culture.

Click chemistries are increasingly used in the design of materials for numerous biomedical applications, including drug delivery, tissue engineering, and controlled cell culture, owing to their selective, efficient, and often cytocompatible reactivities.1-3 Photoclick chemistries that utilize light to trigger or initiate reactions (e.g., azide-alkyne,4 thiol&#8722;ene,5 and tetrazole-alkene6) are of particular interest for the formation or modification of biomaterials. Rapid rates under mild conditions and control of when and where they take place with light make these reactions well-suited for user-directed control of biomaterial properties in the presence of cells.7,8 In particular, thiol&#8722;ene photoclick chemistries have been used to generate hydrogel-based biomaterials with robust mechanical properties5,9 and for the encapsulation of a wide variety of cell types, including, but not limited to, human mesenchymal stem cells (hMSCs), fibroblasts, chondrocytes, and pancreatic cells, with promise for cell culture and delivery.10,11 Further, these chemistries have been used for the spatial patterning of biochemical cues to mimic key aspects of native cell microenvironments and facilitate appropriate cell-matrix interactions, including adhesion, differentiation, and invasion.3,12
For the construction of thiol&#8722;ene hydrogels with light, peptides containing cysteines (thiol) commonly are reacted with polymers functionalized with acrylates or norbornenes (&#39;ene&#39;) for rapid, photoinitiated polymerization under cytocompatible conditions.13 Expanding this toolbox, we sought to establish methods for hydrogel formation with new versatile and accessible building blocks that required minimal synthetic processing or were commercially available toward their broad use as synthetic extracellular matrices.14 Specifically, peptides were modified with allyloxycarbonyl (Alloc)-protected lysines: one for pendant, integrin-binding groups to promote cell adhesion [K(alloc)GWGRGDS = Pep1Alloc] or two for non-degradable or cell-degradable crosslinks [K(alloc)RGKGRKGK(alloc)G or KK(alloc)GGPQGIWGQGK(alloc)K = Pep2Alloc, respectively]. With these sequences, conditions were established for rapid reaction (1-5 min) with four-arm thiol-modified poly(ethylene glycol) (PEG4SH) using cytocompatible doses of long wavelength UV light (10 mW/cm2 at 365 nm) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The resulting hydrogels were stable under cell culture conditions for weeks. To enable cell-driven degradation and remodeling, an enzymatically-cleavable peptide was incorporated within the gel crosslinks (i.e., GPQGIWGQ),15 and a model primary cell, human mesenchymal stem cells (hMSCs), remained highly viable after encapsulation and during culture within these matrices. Further, peptides have been spatially patterned within these materials, and hMSCs remain viable and metabolically active under photopatterning conditions. Alternate pendant peptide sequences, not shown here (e.g., IKVAV, YIGSR, GFOGER, etc.), also may be incorporated within matrices to probe additional cell interactions with the surrounding microenvironment. These results are promising for the application of these hydrogel-based materials for three-dimensional (3D) cell culture and delivery to study and direct cell-matrix interactions for a variety of cell types.
Herein, methods to photoencapsulate cells and subsequently photopattern biochemical cues within the proposed hydrogel system are presented (Figure 1). Techniques to observe and quantify these photopatterns also are demonstrated: notably, i) the quantitative and qualitative use of Ellman&#39;s assay to determine the modification of free thiols within patterned substrates and ii) the complementary qualitative use of fluorescent peptides (AF488Pep1Alloc) to observe these patterns in three dimensions. Further, assays to determine viability (live/dead viability/cytotoxicity staining) and metabolic activity (MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) are presented so that users may determine the cytocompatibility of photoencapsulation and photopatterning conditions for different cell lines within hydrogel matrices. While the protocol is demonstrated for a facile light-based photoclick hydrogel system, the techniques may be applied to numerous other radically-initiated hydrogel systems for photoencapsulation and photopatterning in the presence of cells.

<table><tbody><tr><td>Custom Peptides</td><td>Various Vendors</td><td>----</td><td>Peptides may also be synthesized via standard SPPS techniques with materials from vendors including ChemImpex and ChemPep.</td></tr><tr><td>4-arm PEG Thiol, MW 20k</td><td>JenKem USA</td><td>4ARM-SH</td><td>Listed under Multi-arm Homofunctional PEGs. PEG4SH may also be synthesized as previously referenced.</td></tr><tr><td>Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate</td><td>Colorado Photopolymer Solutions</td><td>Li-TPO</td><td></td></tr><tr><td>Dimethyl Phenylphosphonite</td><td>Sigma Aldrich</td><td>149470</td><td>Caution, causes severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>2,4,6-Trimethylbenzoyl Chloride</td><td>Sigma Aldrich</td><td>682519</td><td>Caution, causes severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Lithium Bromide</td><td>Sigma Aldrich</td><td>213225</td><td></td></tr><tr><td>2-Butanone</td><td>Sigma Aldrich</td><td>360473</td><td></td></tr><tr><td>Flask, Round Bottom, 100&amp;nbsp;ml</td><td>Chemglass</td><td>CG-1506-05</td><td></td></tr><tr><td>80 ml Filter Funnel, Buchner, Medium Frit</td><td>Chemglass</td><td>CG-1402-L-02</td><td>Filter paper inside a regular glass funnel may be used if desired.</td></tr><tr><td>Magnetic Stir Bars</td><td>Various Vendors</td><td>----</td><td></td></tr><tr><td>Magnetic Stirring and Hot Plate</td><td>Various Vendors</td><td>----</td><td></td></tr><tr><td>Vacuum Dessicator</td><td>Various Vendors</td><td>----</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Acros Organics</td><td>16630</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190-250</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Fungizone</td><td>ThermoFisher Scientific</td><td>15290-018</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red</td><td>ThermoFisher Scientific</td><td>15400-054</td><td>Select appropriate medium and trypsin depending on cell type.</td></tr><tr><td>Microcentrifuge tubes</td><td>Various Vendors</td><td>----</td><td>1.5 or 2 ml sizes, sterile.</td></tr><tr><td>BD Syringe with Slip (Luer) Tips (Without Needle)</td><td>Fisher Scientific</td><td>14-823-16H</td><td>Product number listed here is for a 1 ml syringe (16H), Various sizes are available (14-823-XX).</td></tr><tr><td>Fisherfinest Premium Plain Glass Microscope Slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>High-Purity Silicone Rubber, 0.010&quot; Thick, 6&quot; x 8&quot; Sheet, 55A Durometer (Gasket)</td><td>McMaster Carr</td><td>87315K62</td><td></td></tr><tr><td>Ethanol, 200 Proof</td><td>Decon Labs</td><td>2701</td><td></td></tr><tr><td>RainX, Original</td><td>Amazon</td><td>800002243</td><td>May be purchased from other vendors.</td></tr><tr><td>Sigmacote</td><td>Sigma Aldrich</td><td>SL2</td><td></td></tr><tr><td>Photomasks</td><td>Advance Reproductions Corporation</td><td>----</td><td>Photomask Division, different designs may be printed as desired.</td></tr><tr><td>DTNB; Ellman&#039;s Reagent; 5,5-dithio-bis(2-nitrobenzoic acid)</td><td>ThermoFisher Scientific</td><td>PI-22582</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma Aldrich</td><td>E6758</td><td></td></tr><tr><td>Sodium Hydroxide, Pellets/Certified ACS</td><td>Fisher Scientific</td><td>S318</td><td></td></tr><tr><td>Orthophosphoric Acid</td><td>Alfa Aesar</td><td>33266</td><td></td></tr><tr><td>Cysteine Hydrochloride Monohydrate</td><td>Sigma Aldrich</td><td>C7880</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>ThermoFisher Scientific</td><td>L-3224</td><td></td></tr><tr><td>CellTiter 96 Aqueous One Solution Assay</td><td>Promega</td><td>G3582</td><td></td></tr><tr><td>Centrifuge</td><td>Various Vendors</td><td>----</td><td>Capable of speeds at 90-110 x g.</td></tr><tr><td>Multiwell Plate Reader</td><td>Various Vendors</td><td>----</td><td>Capable of reading absorbance at 405 nm in a 96-well plate.</td></tr><tr><td>Epifluorescent or Confocal Microscope</td><td>Various Vendors</td><td>----</td><td>To visualize peptide patterns and cells within hydrogels.</td></tr><tr><td>Omnicure Exfo Series 2000</td><td>Excelitas Technologies</td><td>----</td><td>Alternate light systems may be used to polymerize hydrogels.</td></tr><tr><td>Zeiss Zen Lite Software</td><td>Zeiss</td><td>----</td><td>Available at zeiss.com; compatible with images taken on Zeiss microscopes</td></tr><tr><td>ImageJ</td><td>NIH</td><td>----</td><td>Available at imagej.nih.gov; applicable for general image analysis</td></tr></tbody></table>

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.

The setup and procedure to photoencapsulate cells and subsequently photopattern gels containing encapsulated cells is depicted in Figure 1, and an example for preparing stock solutions to form a 10 wt% gel is provided in Table 1. Using Table 1, the amount of monomers (PEG4SH, Pep2Alloc, &#177;Pep1Alloc) and photoinitiator (LAP) required to polymerize hydrogels is calculated. Based on these calculations, stock solutions of PEG4SH, Pep1Alloc, Pep2Alloc, and LAP are prepared and mixed with and without Pep1Alloc for forming and photopatterning gels, respectively (Figure 1A). Subsequently, cells are collected from plates, counted, and centrifuged in appropriate quantities for encapsulation (Figure 1B). The cell pellet is re-suspended in gel-forming solution (peptide/polymer/LAP in PBS), and cell/monomer mixtures are transferred to glass or syringe molds. Cells are encapsulated within the hydrogel upon application of light (1-5 minutes of 10 mW/cm2 at 365 nm) (Figure 1C). For photopatterning (Figure 1D), gels are soaked with Pep1Alloc and LAP for 30 min to 1 hr and 30 min, allowing diffusion of peptide and initiator into the polymerized matrix. These peptide-laden gels are covered with photomasks with desired patterns and exposed to a second dose of light (1 min) to conjugate the peptides to free thiols within the matrix. Pendant tethers are only covalently linked to the gel in regions exposed to light, facilitating appropriate cell-matrix interactions and mimicking key mechanical and biochemical properties of the native cell microenvironment toward probing cell function and fate in vitro.
Ellman&#39;s assay provides a facile and inexpensive method to quantify gel modification and peptide incorporation within photopatterned substrates. Patterned and non-patterned gels (i.e., gels with or without peptide modification) are soaked in Ellman&#39;s reaction buffer post-polymerization. Next, cysteine standards and the gels soaked in buffer are placed in 48-well plates and incubated with Ellman&#39;s reagent (Figure 2A, left). After 1 hour 30 minutes, aliquots of samples are placed in individual wells of a 96-well plate, and the absorbance is recorded at 405 nm. A calibration curve (Figure 2A, right) from the cysteine standards is plotted (absorbance vs concentration, linear fit), and the quantity of free thiols in gels may be determined based on their dilution factor. These free thiol concentrations for various polymerization and photopatterning conditions of a 10 wt% gel are shown in Figure 2B. Gels polymerized for 1 or 5 min without Pep1Alloc (blue bars, I = 1 min and II = 5 min) have statistically similar free thiol concentrations, indicating that a rapid reaction occurs and gelation is complete within 1 min (two-tailed t-test, p &#62;0.05). Thus, additional exposure to light (2-5 minutes) does not result in further conversion of functional groups. Gels polymerized for 1 min without Pep1Alloc were soaked with Pep1Alloc (3 mg/ml) and LAP (2.2 mM) for 30 and 90 min (green bars, II = 30 min and IV = 90 min) and exposed to a second dose of light for 1 min. The decrease in free thiols (60-80% with respect to the 1 min condition) indicates efficient modification of gels with pendant cues under these conditions. If higher modification is desired, increased concentrations of Pep1Alloc solution can be used as accessibility of Pep1Alloc to free thiols in more dilute peptide solutions may limit conversion; for example, we have found that concentrations up to 20 mg/ml Pep1Alloc produce &#62;90% conversion of free thiols.
Uniquely, patterns of peptides added to the hydrogel may be rapidly imaged with Ellman&#39;s reagent (Figure 3A, under 5 min). However, visualization of the pattern is lost over time (greater than 5 min) owing to diffusion of the yellow TNB2- dianion through the gel. To improve imaging resolution and observe patterns in three dimensions (x-, y-, and z-planes) and in the presence of cells, fluorescent peptide addition (AF488Pep1Alloc) may be utilized. In Figure 3B x-, y- and z-projections of image stacks taken with a confocal microscope are shown, demonstrating pattern resolution (&#181;m scale).
Approximately (94 &#177; 2) % and (94 &#177; 1) % of hMSCs encapsulated within degradable gels (Pep2Alloc = GPQG&#8595;WGQ) remain viable (green cell bodies) 1 and 6 days after encapsulation, respectively, with few dead cells (red nuclei) observed (Figure 4A). Further, hMSC spreading is observed at 6 days post-encapsulation (Figure 4A, inset), indicating that cells can remodel and interact with these MMP-degradable matrices modified with integrin-binding RGDS. Metabolic activity assays performed on cells encapsulated in non-degradable gels (Pep2Alloc = RGKGRK) 1 and 3 days post-encapsulation (Figure 4B) provide a second measure of cell viability and demonstrate that cells remain active for the various photoencapsulation and photopatterning conditions tested with Ellman&#39;s assay (Figure 2B). In particular, there is no significant difference in metabolic activity between 30 min and 90 min incubations with Pep1Alloc+LAP (conditions III and IV) and the initial encapsulation (conditions I and II), indicating that the procedure is appropriate for applications of photopatterning in the presence of encapsulated cells (two-tailed t-test, p &#62; 0.05).
<img alt="Figure 1" src="/files/ftp_upload/54462/54462fig1.jpg" /> 
Figure 1: Setup for encapsulating cells within hydrogels and subsequently photopatterning with biochemical cue. (A) Stock solutions of macromers and photoinitiator are prepared and mixed (PEG4SH = backbone, Pep2Alloc = crosslink, Pep1Alloc = pendant adhesive moiety (RGDS), LAP = photoinitiator). (B) Cells are collected for encapsulation. (C) Cells are mixed with macromer solutions without or with integrin-binding peptides (PEG4SH/Pep2Alloc/LAP or PEG4SH/Pep2Alloc/Pep1Alloc/LAP, respectively) and are encapsulated upon exposure to light. (D) Gels containing excess thiol groups during gel formation (here, PEG4SH/Pep2Alloc/LAP, formed with 2 mM excess thiol) may be patterned with biochemical cues by the subsequent addition of peptides functionalized with a single alloc group (Pep1Alloc, e.g., RGDS, IKVAV, etc.) to promote cell adhesion within specific regions of the gel. Here, patterning of gels with fluorescently-labeled RGDS is shown. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54462/54462fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54462/54462fig2.jpg" /> 
Figure 2: Quantitative Ellman&#39;s assay setup and results to evaluate modification of patterned hydrogels. (A) Gels and cysteine standards are incubated in 48-well plates with Ellman&#39;s reagent. A linear standard curve is plotted to determine cysteine concentration in gel samples. (B) Excess free thiols are incorporated within hydrogels during gel formation and consumed upon patterning with a pendant alloc-peptide (I = 1 min polymerization; II = 5 min polymerization; III = 30 min incubation with Pep1Alloc; IV = 90 min incubation with Pep1Alloc; both III and IV polymerized and patterned with light for 1 min). The data shown illustrate the mean (n = 3) with error bars showing the standard error. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54462/54462fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54462/54462fig3.jpg" /> 
Figure 3: Ellman&#39;s Assay and fluorescent images to visualize photopatterned hydrogels. (A) Ellman&#39;s reagent may be used to rapidly detect patterns (lines) in the x- and y-planes (yellow = unpatterned region, Scale bar = 1 mm). (B) Fluorescent peptides may be used to observe patterns in the x-, y-, and z-planes (green = patterned region; 200 &#181;m scale bar; 10X water-dipping objective; Ex/Em 488/525 nm). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54462/54462fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54462/54462fig4.jpg" /> 
Figure 4: Viability and metabolic activity of cells within non-degradable photopatterned hydrogels. (A) Example confocal z-stack (z-projection; 10X water-dipping objective) of viable (green; Ex/Em 488/525 nm) and dead hMSCs (red; Ex/Em 543/580 nm) encapsulated within hydrogels 24 hr post-encapsulation (Scale bar = 200 &#181;m). Cells spread within these hydrogels 6 days after encapsulation (inset image, Scale bar = 50 &#181;m). (B) Cells are metabolically active 1 and 3 days (dark and light bars, respectively) post-encapsulation for the various polymerization (I = 1 min polymerization; II = 5 min polymerization) and patterning conditions (III = 30 min incubation with Pep1Alloc; IV = 90 min incubation with Pep1Alloc; both polymerized and patterned with light for 1 min). The data shown illustrate the mean (n = 3) with error bars showing the standard error. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54462/54462fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54462/54462tbl1.jpg" /> 
Table 1: Example setup to calculate volumes of stock solutions for making hydrogels. Cells within the spreadsheet that are highlighted blue indicate user-defined parameters; the other quantities are calculated based on these settings. The final volumes for each stock solution to make a gel are highlighted orange. The white cells contain formulas used to calculate the final volumes based on the user-defined parameters. Note that a concentration of 2.2 mM LAP is equivalent to 0.067 wt%.

Watch this Scientific Journal Video about Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications at JoVE.com

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

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 ...

light-mediated-formation-patterning-hydrogels-for-cell-culture

Research

JoVE Journal

Bioengineering

12.9K Views.  University of Delaware. 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. 

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

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 ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

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 ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biology

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 ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

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 ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

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

Summary

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Chapters in this video

Microfabricated Platforms for Mechanically Dynamic Cell Culture

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

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

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

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

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

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy