Name: production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d
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The authors thank T. Palmer and H. Babu (Stanford Neurosurgery) for providing murine NPCs. Vector art in Figure 4 was used and adapted from Servier Medical Art under Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/legalcode). Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-1542152. N.A.S. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health (32GM008412). C.M.M. acknowledges support from an NIH NRSA pre-doctoral fellowship (F31 EB020502) and the Siebel Scholars Program. S.C.H. acknowledges support from the National Institutes of Health (U19 AI116484 and R21 EB018407), National Science Foundation (DMR 1508006), and the California Institute for Regenerative Medicine (RT3-07948). This research received funding from the Alliance for Regenerative Rehabilitation Research &#38; Training (AR3T), which is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institute of Neurological Disorders and Stroke (NINDS), and National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health under Award Number P2CHD086843. The content is solely the responsibility of the authors and does not necessarily represent the views of the National Institutes of Health.

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The authors have nothing to disclose.

Recombinant protein expression and purification is a powerful tool to synthesize biomaterials with high reproducibility. Owing largely to the advent of commercialized molecular cloning, custom recombinant plasmids can be purchased from several suppliers, which significantly reduces the time to work with materials like ELPs. Similarly, plasmids can be requested directly from the originating lab when the original work was supported by a federal contract and the future work will be for non-profit use. The full ELP amino acid sequence has been previously published for several ELP variants31. However, the process from expression to eventual purification of recombinant proteins involves a number of critical steps that can commonly lead to reduced yields or a lower quality product. Some of the most common issues for ELP preparation arise in one of the following: (1) quality of stored bacterial stocks, (2) the first freeze-thaw cycle to disrupt the bacterial membrane, and (3) protein purification through thermal cycling.
A major difference between protein expression and other non-biological means of producing materials is that we are leveraging the biological machinery of recombinant hosts to synthesize the polymers. Subsequently, this technique comes with a unique limitation: cellular death or damage. Cell death most commonly manifests itself as a reduced number of bacterial colonies after streaking a plate or abnormally small colonies that grow relatively slowly. Bacterial stocks, if maintained carefully, can remain stable for years; however, successive freeze-thaw cycles due to repeated use or freezer failure can reduce cell viability or lead to DNA damage. Typical BL21 bacteria stocks use between 10% and 40% glycerol by volume mixed with suspended cells. The purpose of the glycerol is to reduce membrane damage from nucleating ice crystals during freezing. Therefore, using low concentrations (&#60;10%) can lead to a compromised membrane, while higher concentrations (&#62;40%) can suppress the freezing point sufficiently to where the stock never freezes leading to cell death. However, even within optimal glycerol levels, bacterial stocks should not be allowed to fully thaw as a combination of membrane damage from re-freezing and cytotoxic effects from the glycerol can lead to reduced stock viability and DNA damage. Therefore, if it is observed that a bacterial stock results in a low colony count or that the cells are dividing at a consistently slow rate (manifested as a slow OD600 ramp rate during expression), re-transforming the plasmid and making a new stock is a simple first approach to troubleshoot this problem. With this in mind, to ensure the long-term upkeep of bacterial stocks and integrity of DNA, it is best to store the copies of your plasmid as purified DNA frozen in water and not within cells. Storing DNA in this way will ensure that in unforeseen events such as a failed stock or freezer failure, a reliable source of the original DNA can be used for transformation.
Another critical step in ELP fabrication is the purification of the target protein from the expression host. Protein extraction from E. coli is achieved by breaking the cell wall using nucleating ice crystals that form throughout the suspended cell lysate upon freezing, which is further compounded with successive freeze-thaw cycles. Alternative methods for rupturing the cell wall can be utilized such as sonication or a press. In particular, consecutive freeze-thawing of the lysate is advantageous as it only requires a freezer and no other specialty equipment. However, this procedure nonspecifically releases DNA, RNA, and protein contaminants, in addition to proteases that have the potential to degrade the target protein. Therefore, to avoid contamination and reduced yield, deoxyribonuclease I (DNase) and phenylmethanesulfonyl fluoride (PMSF) are added to the cell lysate to degrade the DNA and inhibit proteases, respectively. The presence of DNA prior to the addition of DNase can be observed visually as a &#39;stringy&#39; appearance throughout the re-suspended cell lysate following the first thaw. DNase actively degrades this DNA and thus reduces the viscosity of the cell lysate making it easier to purify via centrifugation. Optimal break down of DNA can be visually confirmed by ensuring that the cell lysate appears to be entirely liquid and that the stringy appearance is no longer visible. We have observed in practice that the addition of ~0.1 mg of DNase per mL cell lysate is sufficient to achieve necessary degradation. However, if the presence of DNA is still observed, more DNase can be added followed by an additional two to three hours of agitation. A similar issue can also arise if DNase is added prematurely before any of the lysate has had the potential to sufficiently thaw. In this case, the colder temperatures can limit the efficiency of DNA degradation due to the premature inactivation of DNase. To avoid this issue, it is often best practice to allow the re-suspended pellet to thaw for approximately 8 hours prior to treatment with DNase. In addition, if low protein yields are reported and the breakdown of DNA has been sufficient, the addition of more PMSF to help further reduce potential protein degradation from proteases may be required.
Additional considerations for ensuring optimal expression of ELPs include a careful understanding of the benefits and limitations of a chosen antibiotic. Here, pET15b vectors containing an ampicillin resistance gene were used for protein expression. Functionally, the pET vector series allow for significant protein expression with as much as 50% of a bacterium&#39;s protein expression dedicated to the target protein following a successful induction32,33. However, ampicillin as a selection antibiotic comes with some limitations that may interfere with optimum expression. First, degradation of ampicillin in the presence of E. coli can occur rapidly due to the release of beta-lactamase. If a sufficient quantity of the ampicillin is degraded, the ampicillin-encoding plasmid (i.e. the ELP-encoding plasmid) may be lost entirely. As a result, when expressing ELPs for longer durations, protein expression levels should be carefully monitored at successive time points to ensure sufficient amount of the ELP-encoding gene remains to allow for desirable expression. Possible methods for troubleshooting the buildup of beta-lactamases include spinning down the starter culture and re-suspending the cells in antibiotic-free medium prior to inoculating the expression medium. This process effectively limits the transfer of antibiotic-degrading enzymes and ensures a greater portion of the cells contain the target-protein-encoding vector. Additionally, ampicillin has a limited shelf life of approximately two to three weeks. Therefore, culture plates for protein expression should be stored at 4 &#176;C for a maximum of two weeks prior to use. Finally, to ensure the efficacy of ampicillin within the starter and expression media, the ampicillin stock solution should be produced fresh immediately before use, as long-term storage may lead to a less effective antibiotic.
The presence of an LCST allows for the simple purification of ELPs through thermal cycling. Specifically, at a higher temperature and in the presence of salts, entropic forces cause ELPs to become less soluble and subsequently form a polymer-rich coacervate phase. On the other hand, at lower temperatures, ELPs remain soluble and readily dissolve into the solution. Cycling between these two temperature regimes coupled with centrifugation steps to collect and discard the non-ELP-containing phase successively concentrates the protein and simultaneously reduces the existence of non-ELP contaminants.
However, there are a number of stages where ELPs can be lost in this purification process. First, prior to every cold spin, the protein-containing solution is alkalized to a pH of 9.0. This higher pH serves to deprotonate certain amino acids on the protein backbone, effectively leaving them in a charged state and further enhancing their solubility. Consequently, foregoing this step or not allowing sufficient time for protein dissolution can lead to a reduction in yield as non-solubilized proteins will be pelleted during centrifugation and discarded.
Similarly, target proteins can be lost during the hot spin procedure when the ELP is pelleted. Initially, NaCl is added to the protein-rich supernatant to reduce the solubility of the ELP. The salts work to shield electrostatic interactions between the protein and water molecules, causing the protein to separate from the aqueous phase. This effect is amplified by heating the solution, which, due to entropic effects, further breaks down the hydrous &#39;cage&#39; surrounding ELPs and forces the aggregation of the proteins. At lower protein concentrations (i.e., the first thermal cycle), the addition of salts alone is often insufficient to cause this phase separation. However, as the concentration of protein increases (i.e., later thermal cycles), and there are less secondary contaminants to interact with the salts, the ELP will more readily precipitate. As a result, if salts are added too quickly, they may become physically trapped by aggregating proteins, which effectively reduces the salt concentration of the solution and limits further protein precipitation. Thus, the salt should be added in three small batches to ensure they have sufficient time to homogenously distribute through the solution. As a final note, variations to the ELP backbone, either through further modifications to the guest residue of the elastin-like region or changes to the bio-active region can significantly impact the LCST behavior. Consequently, to ensure optimal protein yields across protein variants, it is crucial to optimize the pH, salt concentration, and salt type (e.g., monovalent or divalent) for the cold and hot spins.
Running SDS-PAGE upon protocol completion is recommended as it can be used to easily determine if significant ELP loss occurs during any of the purification steps. Briefly, if ELP is detected in the supernatant following a hot spin, then the protein is not being effectively precipitated. Similarly, if ELPs are identified in a sample of solubilized pellet following a cold spin, then the protein is not being effectively dissolved.
ELP hydrogels offer many advantages over synthetic or naturally-derived materials. Specifically, the use of the amine-reactive crosslinker THPC affords a low-cost, simple, and tunable mechanism of protein crosslinking. However, there are distinct limitations within the crosslinking protocol that should be noted. THPC is oxygen sensitive, and if stored under improper conditions, it can quickly deteriorate in reaction efficiency. In addition, due to its reactivity with primary amines, THPC may react with surrounding proteins in media or those on the cell surface that are rich in amines. Therefore, when forming ELP hydrogels, it is recommended to avoid media contamination with the cell pellet to reduce possible exogenous protein cross-reactivity and thus, a reduction in crosslinking efficiency. Finally, this crosslinking mechanism precludes the bio-active region sequence to those containing no lysine residues and thus, limits potential integration of some cell-adhesive motifs (e.g., IKVAV34). To address these limitations, modifications to the ELP backbone with azide and bicyclononyne (BCN) reaction partners allows for bio-orthogonal crosslinking, as previously described27.
It should be noted that the ELP LCST behavior plays an important role in dictating hydrogel microstructure. At temperature regimes above the LCST, ELPs precipitate out of solution leading to the formation of protein-rich and protein-deficient phases that can influence matrix porosity and crosslinking efficiency of the matrix9. Because most cell culture experiments are conducted at physiologically relevant temperatures (~37 &#176;C) above the ELP LCST, these effects should be considered. For the hydrogels to effectively crosslink and form an interconnected protein network, the primary amine from the lysine must be physically accessible to the THPC crosslinker. If the ELP aggregation occurs before reaching sufficient crosslinking, ELPs trapped within the protein-rich phase may be inaccessible and thus unable to participate in crosslinking. To address this limitation, our protocol requires an initial 15 min crosslinking period at room temperature, which allows for preliminary crosslinking of the hydrogel before the ELP undergoes its thermal phase transition. This room temperature incubation is followed by an additional 15 min incubation at 37 &#176;C to finalize hydrogel crosslinking. This procedure is critical for sufficient crosslinking and robust, reproducible gelation of the ELP material.
In conclusion, recombinant protein hydrogels fabricated using ELP offer exceptional tunability of the protein sequence and therefore the 3D cell microenvironment. ELP polymers have been shown to be expressible in high yields, easily purified owing to their LCST behavior, and biocompatible in a wide variety of in vitro and in vivo systems. The use of E. coli as a recombinant host provides a simple and inexpensive procedure that gives rise to near perfect control of polymer molecular weight and functionality. In conjunction, this technique allows for robust tunability and reproducibility of the hydrogel platform allowing for the culture of a wide range of cell types in 3D. Finally, this ELP hydrogel platform is amenable to many downstream biochemical assays including qRT-PCR, Western blot, DNA extraction, and cell immunostaining9.

Over the past century, two-dimensional (2D) tissue culture has developed into an integral toolset for studying fundamental cell biology in vitro. In addition, the relatively low-cost and simple protocols for 2D cell culture have led to its adoption across many biological and medical disciplines. However, past research has shown that traditional 2D platforms can lead to results that deviate markedly from those collected in vivo, causing precious time and funding wasted for clinically oriented research1,2,3. We and others hypothesize that this discrepancy may be attributed to the lack of native biochemical and biophysical cues provided to the cells cultured on 2D surfaces, which can be necessary for optimal proliferation and maturation of various cell types.
To address these limitations and help bridge the gap between 2D in vitro and in vivo studies, researchers have developed three-dimensional (3D) hydrogel platforms for cell-encapsulation1,4,5,6. Hydrogels are ideal materials to recapitulate the endogenous microenvironment of the extracellular matrix (ECM) in vivo due to their tissue-like mechanical properties and water-swollen structure that enables rapid transport of nutrients and signaling factors7,8. Furthermore, 3D hydrogels can be designed to have independent control over the mechanical and biochemical properties of the scaffold. Both matrix mechanics9,10,11,12 and cell-adhesive ligands13,14,15 are well-known to influence cell behavior in vitro and in vivo. Thus, 3D hydrogels with tunable properties offer a platform to study the causal relationships between cells and their microenvironment. Criteria for an ideal 3D hydrogel matrix include simple, non-cytotoxic cell-encapsulation as well as independent tunability of physiologically relevant mechanical properties and mimics of native cell-adhesive motifs.
Both synthetic (e.g., polyethylene glycol, polylactic acid, poly(glycolic acid)) and naturally-derived (e.g., alginate, collagen, Matrigel) hydrogels have advantages over 2D in vitro culture platforms; however, they also have significant shortcomings which limit their applicability. First, many synthetic and naturally-derived platforms require harsh crosslinking conditions that can be potentially toxic to mammalian cells, leading to decreased cell viability7. Additionally, many synthetic platforms lack native bioactivity and need to be functionalized through secondary chemical reactions, which can add increased cost and complexity16. Finally, while naturally-derived materials typically contain intrinsic bio-active domains, they are often plagued by high batch-to-batch variability and often are limited to forming relatively weak gels7,17.
Recombinant protein engineering presents a unique toolset for materials design by allowing explicit control over protein sequence and, by extension, the potential mechanical and biochemical properties of the final hydrogel scaffold18. Additionally, by leveraging the well-known biological machinery of Escherichia coli (E. coli) to express proteins, materials can be produced cost-effectively and consistently with limited inter- and intra-batch variability. The elastin-like protein (ELP) presented here has three engineered domains: (1) a T7 and His6 tag that allows for labeling via fluorescently tagged antibodies, (2) an &#39;elastin-like&#39; region that confers elastic mechanical properties and allows for chemical crosslinking, and (3) a &#39;bio-active&#39; region that encodes for cell-adhesive motifs.
Our elastin-like region is based on the canonical (Val-Pro-Gly-Xaa-Gly)5 elastin sequence where four of the &#39;Xaa&#39; amino acid sites are isoleucine (Ile), but could be designed to be any amino acid except proline. This sequence endows recombinant ELPs with lower critical solution temperature (LCST) behavior that can be exploited for simple purification post-expression via thermal cycling19,20. This LCST property can be tuned to thermally aggregate at different temperatures by modifying the guest &#39;Xaa&#39; residue21,22.
Here, the &#39;Xaa&#39; position on one of the five elastin-like repeats has been replaced with the amine-presenting lysine (Lys) amino acid, which is utilized for hydrogel crosslinking. Our previous work has shown non-cytotoxic and robust crosslinking via reaction with the amine-reactive crosslinker tetrakis(hydroxymethyl)phosphonium chloride (THPC)23. By varying overall protein content and crosslinker concentration, we are able to produce hydrogels that can be tuned to span a physiologically relevant stiffness range (~0.5-50 kPa)9,23,24. In addition to tuning mechanical properties, cell adhesion within the hydrogel results from the integration of canonical cell-adhesive domains within the backbone of the ELP protein. For example, the incorporation of the extended fibronectin-derived &#39;RGDS&#39; amino acid sequence allows for cell adhesion and conformational flexibility, while the scrambled, non-binding &#39;RDGS&#39; variant restricts cell-matrix adhesion24. By modulating the ratio of cell-adhesive to non-adhesive proteins as well as the total protein concentration, we are able to effectively produce hydrogels which span a wide range of ligand concentration. Resultantly, we have developed a hydrogel platform with decoupled biochemical and biophysical properties, which can be independently tuned for optimal 3D culture of various cell types.
In addition to matrix stiffness and adhesive ligand tunability, recombinant hydrogels offer the capability to design specific material degradation profiles, which is necessary for cell spreading, proliferation, and migration within a 3D context4,9. This degradation is afforded by cell secretion of proteases that specifically target either the extended &#39;RGDS&#39;9 or elastin-like sequence25. ELP hydrogels have also been shown to support the subsequent biochemical assays that are necessary for studying cell viability and function including immunocytochemistry as well as DNA/RNA/protein extraction for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot9. ELP variants have also been used in a number of in vivo models and are known to be well tolerated by the immune system26.
Taken together, ELP as a material platform for cell-encapsulation studies boasts a wide variety of benefits compared to synthetic or naturally-derived material platforms, which often lack the same degree of biochemical and biophysical tunability and reproducibility. Additionally, ELP&#39;s simple and non-cytotoxic use with a wide variety of cell types (e.g., chick dorsal root ganglia14,24, murine neural progenitor cells9, human mesenchymal stem cells27, bovine neonatal chondrocytes28, human endothelial cells29,30) allows for a more physiologically relevant model of the endogenous 3D ECM compared to 2D cell culture. Herein, we present a protocol for the expression of recombinantly-derived, ELPs for the use as a tunable hydrogel platform for 3D cell encapsulation. We further present the methodology for down-stream fluorescent labeling and confocal microscopy of encapsulated cells.

<table><tbody><tr><td>&lt;strong&gt;Elastin-Like Protein Expression and Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm Petri Dishes</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>70% Ethanol</td><td>RICCA Chemical</td><td>2546.70-1</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Sigma-Aldrich</td><td>A3920-500G</td><td></td></tr><tr><td>Ampicillin</td><td>Thermo Fisher Scientific</td><td>BP1760-25G</td><td></td></tr><tr><td>Bacto Agar</td><td>Thermo Fisher Scientific</td><td>9002-18-0&amp;nbsp;</td><td></td></tr><tr><td>BL21(DE3)pLysS Competent Cells</td><td>Invitrogen</td><td>C606003</td><td></td></tr><tr><td>Chloramphenicol</td><td>Amresco</td><td>0230-100G&amp;nbsp;</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>EDTA disodium salt, dihydrate</td><td>Thermo Fisher Scientific</td><td>O2793-500</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific</td><td>A451-4</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>BP1755-10G</td><td></td></tr><tr><td>Luria Broth</td><td>EMD Millipore</td><td>1.10285.5007</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MP Biomedicals</td><td>195381</td><td></td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S 8045-1KG</td><td></td></tr><tr><td>Syringe Filter Unit (0.22 &amp;mu;m)</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr><tr><td>Terrific Broth</td><td>Millipore</td><td>71754-4</td><td></td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>&lt;strong&gt;Cell Encapsulation in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Millipore</td><td>SLGV004SL</td><td></td></tr><tr><td>0.5 mm thick silicone sheet</td><td>Electron Microscopy Science</td><td>70338-05</td><td></td></tr><tr><td>24-well tissue culture plates&amp;nbsp;</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Disposable Biopsy Punch (2 mm)</td><td>Integra Miltex</td><td>33-31</td><td></td></tr><tr><td>Disposable Biopsy Punch (4 mm)</td><td>Integra Miltex</td><td>33-34</td><td></td></tr><tr><td>Disposable Biopsy Punch (5 mm)</td><td>Integra Miltex</td><td>33-35</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>No. 1 12 mm glass coverslips</td><td>Thermo Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td><td>Sigma-Aldrich</td><td>404861-100ML</td><td></td></tr><tr><td>0.5% Tryspin/EDTA</td><td>Thermo Fisher&amp;nbsp;</td><td>15400054</td><td></td></tr><tr><td>&lt;strong&gt;Immunocytochemistry of Cells in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>16% (w/v) Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15701</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Molecular Probes</td><td>D1306&amp;nbsp;</td><td></td></tr><tr><td>Donkey Serum</td><td>Lampire Biological Labs</td><td>7332100</td><td></td></tr><tr><td>Goat anti-mouse Secondary Antibody (AF488)</td><td>Molecular Probes</td><td>A-11017</td><td></td></tr><tr><td>Goat anti-rabbit Secondary Antibody (AF546)</td><td>Molecular Probes</td><td>A-11071</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-072</td><td></td></tr><tr><td>Mouse Nestin Primary Antibody</td><td>BD Pharmingen</td><td>556309</td><td></td></tr><tr><td>Mouse Sox2 Primary Antibody</td><td>Cell Signaling Technology</td><td>23064S</td><td></td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Vectashield Hardset Mounting Medium&amp;nbsp;</td><td>Vector Labs</td><td>H-1400&amp;nbsp;</td><td></td></tr></tbody></table>

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.

The ELPs used in this protocol are comprised of five regions: a T7 tag, His6 tag, enterokinase (EK) cleavage site, a bio-active region, and an elastin-like region (Figure 1). The T7 and His6 tags allow for easy identification through standard Western blot techniques. Introduction of the EK cleavage site allows for the enzymatic removal of the tag region, if needed. The bio-active region encodes for the extended, fibronectin-derived cell-adhesive (&#39;RGDS&#39;) or non-adhesive (&#39;RDGS&#39;) sequences. Lastly, the central repeat of the elastin-like region contains a lysine group at the guest residue site which enables crosslinking via THPC while the flanking repeats contain isoleucine to achieve an LCST of ~32 &#176;C31.
Post expression, SDS-PAGE or Western blot can be used to visualize the molecular weight and confirm the identity of ELPs that contain antibody tags, such as T7 (MASMTGGQQMG) or His6 (HHHHHH) (Figure 2). Successful expression under controlled conditions yields a highly homogeneous product represented by the presence of a single dark band at the approximate molecular weight (~37 kDa) of these proteins using both SDS-PAGE (Figure 2A) and Western blot (Figure 2B).
In uncontrolled conditions, the presence of lower molecular weight bands on a Western blot suggests that some fraction of the proteins was not completely translated and/or were degraded after expression (Figure 2C). Specifically, the masses here are evenly spaced by ~9 kDa which roughly corresponds to the weight of one bio-active region and three elastin-like regions (a &#39;repeat&#39;), or approximately a fourth of the target protein. These smaller protein fragments are usually present when the expression is carried out at a higher temperature (&#62;32 &#176;C) as in Figure 2C. The presence of these lower molecular weight proteins can lead to unpredictable mechanical properties. Thus, regular screening post expression is recommended to ensure a high quality final product.
The mechanical stiffness of ELP-based hydrogels can be modified by manipulating the concentration of ELP or the ratio of THPC reactive groups:ELP primary amines. Concurrently, the concentration of cell-adhesive ligands can be tuned by changing the ratio of ELP variants with the cell-adhesive (RGDS) to non-adhesive (RDGS) sequences within any stiffness regime. By manipulating these two variables, we can produce gels that have a spectrum of mechanical properties and ligand concentrations (Figure 3).
To encapsulate cells within 3D ELP hydrogels, the desired number of cells are suspended in the medium and centrifuged to produce a cell pellet (Figure 4A). The medium is aspirated from the tube, and the cells are re-suspended uniformly in the ELP solution of the desired concentration. Next, THPC solution is added to the cell/ELP suspension and pipetted thoroughly to form a homogenous mixture. This solution is quickly transferred to sterile silicone molds within a 24-well plate using a pipette and allowed to crosslink at room temperature and 37 &#176;C for 15 min each (Figure 4B). Finally, the medium is added to the well plate and incubated at 37 &#176;C in the experiment.
Live/dead staining can be used to assess cell viability and successful cell encapsulation within ELP hydrogels. As illustrated in Figure 5, adult murine neural progenitor cells (NPCs) show high cell viability over 7 days within a 3% (w/v) ELP hydrogel.
3D ELP hydrogels have been previously shown to support NPC stem maintenance measured through the expression of canonical NPC protein markers SRY (sex determining region Y)-box 2 (Sox2) and nestin9. NPCs encapsulated in 3% (w/v) ELP hydrogels with low THPC crosslinking show high expression of nuclear-localized Sox2 and cytoplasmic nestin filaments via immunostaining and confocal imaging (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57739/57739fig1.jpg" /> 
Figure 1: A schematic representation of the ELP and corresponding amino acid sequences. The ELP used in this study contains a T7 and His6 tag for antibody-based imaging, a bioactive region for introduction of cell-adhesive domains, and an elastin-like region that confers elastic mechanical properties and allows for chemical crosslinking. <a href="/files/ftp_upload/57739/57739fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57739/57739fig2.jpg" /> 
Figure 2: Target protein expression can be validated with SDS-PAGE and Western blot to confirm the molecular weight and identity of the final lyophilized product. Pure full-length ELP runs at a molecular weight of 37 kDa as reported by both SDS-PAGE (A) and Western blot using the T7 (MASMTGGQQMG) or histidine 6 (HHHHHH) tag (B). (C). Impure batches of ELP due to the deviations in the ELP expression protocol can lead to the expression of ELPs with lower molecular weights. <a href="/files/ftp_upload/57739/57739fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57739/57739fig3.jpg" /> 
Figure 3: RGDS ligand content can be independently tuned from mechanical properties within ELP hydrogels. 5% (w/v) and 3% (w/v) ELP hydrogels have shear moduli of ~800 Pa and ~400 Pa, respectively. Hydrogels with a 1:1 ratio of THPC reactive groups:ELP primary amines were crosslinked at room temperature for 15 min, heated to 37 &#176;C, and allowed to equilibrate for 5 min prior to measurement. Data are mean &#177; s.d., ***p &#60;0.001. <a href="/files/ftp_upload/57739/57739fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57739/57739fig4.jpg" /> 
Figure 4: Schematic of cell encapsulation in ELP hydrogels. (A). Cells are initially dissociated into a single-cell suspension in the medium and pelleted using a centrifuge. The medium is aspirated from the tube, and the cells are re-suspended in ELP solution at the desired concentration and mixed well. Finally, the THPC crosslinking solution is added and mixed well. (B). Immediately following the addition of THPC, the solution is cast into a silicone mold with a pipette. The solution is allowed to crosslink at room temperature for 15 min followed by a second 15 min incubation at 37 &#176;C. The medium is then added to the culture well for the duration of the experiment. <a href="/files/ftp_upload/57739/57739fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57739/57739fig5.jpg" /> 
Figure 5: Neural progenitor cells maintain high viability in ELP hydrogels. Representative image of neural progenitor cells encapsulated in 3% (w/v) ELP hydrogels with 1:1 crosslinking (THPC reactive groups:ELP primary amines) after 7 days in culture. Green: live staining (calcein-AM); Red: dead staining (ethidium homodimer). Scale Bar = 100 &#181;m.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57739/57739fig6.jpg" /> 
Figure 6: 3D ELP hydrogels support neural progenitor cell stem maker expression. Immunofluorescence image of neural progenitor cells expressing Sox2 and nestin proteins after 7 days of culture in ELP hydrogels. Images show cells encapsulated in 3% (w/v) ELP gels with 0.5:1 crosslinking (THPC reactive groups:ELP primary amines). Blue: DAPI (nuclei); Red: Sox2; Green: nestin. Scale Bar = 25 &#181;m. <a href="/files/ftp_upload/57739/57739fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

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

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 ...

production-elastin-like-protein-hydrogels-for-encapsulation

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.3K Views.  Stanford University. Recombinant eiwit-engineered hydrogels zijn voordelig voor 3D-celkweek als ze toe voor volledige tunability van de ruggengraat van het polymeer, en dus de cel communicatie. Hier beschrijven we het proces van recombinante elastine-achtige EiwitReiniging en de toepassing ervan in 3D hydrogel cel inkapseling.

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

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

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

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

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

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

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

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Chemistry

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

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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 ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

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

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