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 border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Elastin-Like Protein Expression and Purification</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm Petri Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">FB0875713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>RICCA Chemical</td>
			<td style="width:123px;">2546.70-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">A3920-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP1760-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">9002-18-0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21(DE3)pLysS Competent Cells</td>
			<td>Invitrogen</td>
			<td style="width:123px;">C606003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloramphenicol</td>
			<td style="width:260px;">Amresco</td>
			<td style="width:123px;">0230-100G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA disodium salt, dihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">O2793-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">A451-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP1755-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Broth</td>
			<td style="width:260px;">EMD Millipore</td>
			<td>1.10285.5007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>VWR</td>
			<td style="width:123px;">52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>MP Biomedicals</td>
			<td style="width:123px;">195381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">S 8045-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Filter Unit (0.22 &#956;m)</td>
			<td>Millipore</td>
			<td style="width:123px;">SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terrific Broth</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:123px;">71754-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Encapsulation in 3D ELP Hydrogels</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#956;m syringe filters</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:123px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mm thick silicone sheet</td>
			<td>Electron Microscopy Science</td>
			<td style="width:123px;">70338-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well tissue culture plates&#160;</td>
			<td>Corning</td>
			<td style="width:123px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (2 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (4 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-34</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (5 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)&#160;</td>
			<td>Corning</td>
			<td style="width:123px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">No. 1 12 mm glass coverslips</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">12-545-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">404861-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5% Tryspin/EDTA</td>
			<td>Thermo Fisher&#160;</td>
			<td style="width:123px;">15400054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Immunocytochemistry of Cells in 3D ELP Hydrogels</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% (w/v) Paraformaldehyde (PFA)</td>
			<td style="width:260px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">15701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:260px;">Roche</td>
			<td style="width:123px;">3116956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Molecular Probes</td>
			<td style="width:123px;">D1306&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey Serum</td>
			<td style="width:260px;">Lampire Biological Labs</td>
			<td style="width:123px;">7332100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse Secondary Antibody (AF488)</td>
			<td style="width:260px;">Molecular Probes</td>
			<td style="width:123px;">A-11017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit Secondary Antibody (AF546)</td>
			<td style="width:260px;">Molecular Probes</td>
			<td style="width:123px;">A-11071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum</td>
			<td>Gibco</td>
			<td style="width:123px;">16210-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Mouse Nestin Primary Antibody</td>
			<td style="width:260px;">BD Pharmingen</td>
			<td style="width:123px;">556309</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Sox2 Primary Antibody</td>
			<td style="width:260px;">Cell Signaling Technology</td>
			<td style="width:123px;">23064S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Nail Polish</td>
			<td style="width:260px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">72180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">X100-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield Hardset Mounting Medium&#160;</td>
			<td style="width:260px;">Vector Labs</td>
			<td style="width:123px;">H-1400&#160;</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

12.2K Views.  Stanford University. 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.

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

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

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 ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

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

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

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

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

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

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

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

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

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...