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.

1. ELP Expression Protocol
<ol>
	<li>Day 1: Growing the starter colony
	<ol>
		<li>Prepare ampicillin and chloramphenicol agar plates by autoclaving 25 g of Luria broth and 15 g of agar per 1 L of ultrapure water. Once the solution has cooled to ~60 &#176;C, add 1 mL of ampicillin stock (100 mg/mL in ultrapure water) and 1 mL of chloramphenicol stock (34 mg/mL in 70% ethanol) to 1 L of agar solution for final concentrations of 100 &#181;g/mL and 34 &#181;g/mL, respectively. Transfer 20 mL of final solu...

<ol>
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E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+recombinant+proteins+by+fusion+with+thermally-responsive+polypeptides.">Purification of recombinant proteins by fusion with thermally-responsive polypeptides.</a> Nature Biotechnology. 17 (11), 1112-1115 (1999).</li><li>Aladini, F., Araman, C., Becker, C. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+characterization+of+elastin-like+polypeptides+(ELPs)+with+variable+guest+residues.">Chemical synthesis and characterization of elastin-like polypeptides (ELPs) with variable guest residues.</a> Journal of Peptide Science. 22 (5), 334-342 (2016).</li><li>McMillan, R. A., Caran, K. L., Apkarian, R. P., Conticello, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Topographic+Imaging+of+Environmentally+Responsive,+Elastin-Mimetic+Hydrogels.">High-Resolution Topographic Imaging of Environmentally Responsive, Elastin-Mimetic Hydrogels.</a> Macromolecules. 32 (26), 9067-9070 (1999).</li><li>McMillan, R. A., Conticello, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+Elastin-Mimetic+Protein+Gels+Derived+from+a+Well-Defined+Polypeptide+Precursor.">Synthesis and Characterization of Elastin-Mimetic Protein Gels Derived from a Well-Defined Polypeptide Precursor.</a> Macromolecules. 33 (13), 4809-4821 (2000).</li><li>Chung, C., Lampe, K. J., Heilshorn, S. 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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 aci...

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&#3...

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 ...

The overall goal of this protocol is to express and purify recombinantly derived elastin-like proteins for use as a tunable hydrogel for 3D cell encapsulation. Additionally, this protocol presents the methodology for downstream fluorescent labeling in confocal microscopy of encapsulated cells. This method can help answer key questions within the fields of biomaterials, regenerative medicine, and cell biology, such as understanding the role of cell-extracellular matrix interactions in 3D.The main advantage of this technique is that it provides a reproducible, tunable, and modular material platform that is amenable to the study of multiple cell types. Overall, the implementation of this technique can improve our understanding of the critical cell ECM signaling parameters that may dictate the success of future cell-based regenerative therapies. Streak an ampicillin and chloramphenicol agar plate with a premade bacterial stock containing a vector that encodes for the ELP of interest.Then incubate the streaked plate upside down at 37 degrees Celsius overnight to allow for bacterial growth. The following day, pick a single colony from the plate and inoculate the prewarmed starter culture. Incubate the culture in a shaker at 37 degrees Celsius for 16 hours.After the incubation period, use a serological pipette to transfer 20 milliliters of the starter culture to each expression flask. Leave the expression flask shaking for one hour. After one hour, measure the optical density at 600 nanometers of an aliquot from the expression flask.As soon as the reading reaches 0.6, reduce the temperature of the shaker to 32 degrees Celsius. When the reading reaches 0.8, add one milliliter of one molar sterile filtered IPTG to induce ELP expression in the expression flask. Allow the culture to express for seven hours.After seven hours, centrifuge the expression media at 12, 000 x g for 15 minutes at four degrees Celsius in a floor centrifuge. After the centrifugation, use a spatula to collect the cell pellet from the centrifuge container and place into a preweighed ziploc bag. Next, dissolve the cell pellet in sterile filtered TEN buffer.Massage the cell pellet to form a homogenous solution. Freeze the ziploc bag with resuspended cell pellet in a secondary container at 80 degrees Celsius. To the thawed cell pellet, add approximately 30 to 40 milligrams of deoxyribonuclease and one milliliter of 100 millimolar PMSF for every 100 milliliters of cell lysate.Incubate the lysate at four degrees Celsius on a shaker overnight. Following the third freeze-thaw cycle, centrifuge the samples at at least 15, 000 x g for one hour at four degrees Celsius. Post centrifugation, pour off the supernatant from the centrifuge container and transfer the solution to a new centrifuge container.To every 100 milliliters of supernatant, add 5.84 grams of sodium chloride in three parts to a final one molar concentration. Incubate the suspension in a shaking incubator at 37 degrees Celsius for three hours. Following the incubation, centrifuge the samples at at least 15, 000 x g for one hour at 37 degrees Celsius.Post centrifugation, discard the supernatant from the centrifuge container and mass the pellet. Next, add 10 milliliters of autoclaved ultrapure water per gram of pellet obtained after centrifugation. To ensure that the protein dissolves completely, use a metal spatula to mash and resuspend the pellet.Incubate the resuspended pellet at four degrees Celsius overnight on an orbital shaker. Repeat the cold and hot centrifugation steps for a total of three cycles to further purify the ELP. Next, dialize the residual supernatant in a 3.5 kilodalton dialysis membrane against four liters of prechilled ultrapure water at four degrees Celsius for three days to desalt the protein solution.Centrifuge the final dialyzed solution in a precooled centrifuge. Freeze the supernatant at 80 degrees Celsius in a preweighed conical tube. Finally, lyophilize the frozen solution for three days and then weigh the final lyophilized product to determine protein yield.With the help of a biopsy punch, create holes in a 0.5 millimeter thick silicone sheet. Then cut a square around each hole. Next, use tweezers to remove the plastic wrap on each side of the individual molds.After removing the plastic wrap, use the tweezers to position the identical number of bare silicone molds and glass cover slips in alternating rows on the lid of a 48 well plate for subsequent plasma bonding. After all cell culture materials have been fabricated, the preparation of the cell type of interest for downstream hydrogel encapsulation may begin. After dissociating the cells into a single cell suspension, aliquot the desired number of cells into a 1.5 milliliter centrifuge tube.Then, centrifuge the cells at approximately 200 x g for three minutes. Post centrifugation, aspirate the supernatant and store the pellet on ice. Resuspend the cell pellet in ELP stock solution to 80%of the final volume.Then use a pipette to mix the solution into a homogenous suspension of cells in ELP. For the remaining 20%final volume, add THPC stock solution to the cell ELP suspension. Pipette several times to ensure the formation of a homogenous mixture, while avoiding the formation of air bubbles.Next, in a circular motion, pipette the cell ELP THPC mixture into each mold in a 24 well plate. Then incubate the samples at room temperature for 15 minutes. After 15 minutes, incubate the samples at 37 degrees Celsius for an additional 15 minutes.Once the incubation is over, carefully add 750 microliters of warm cell culture medium to each well in a 24 well plate. Leave the hydrogel at 37 degrees Celsius for the desired culture duration. First, aspirate the medium from each well.Then wash the wells with one milliliter of warm DPBS. After washing is completed, add 750 microliters of fixation solution to each well. Then, incubate the 24 well plate at 37 degrees Celsius for 30 minutes.After the incubation, aspirate the fixation solution from each of the wells and discard the fixative in a hazardous waste container. Next, add one milliliter of DPBS to the sample and immediately discard the DPBS into a hazardous waste container. Next, add 750 microliters of permeabilization solution to permeabilize the sample for one hour at room temperature on the rocker at 15 revolutions per minute.After one hour, aspirate the permeabilization solution and add 750 microliters of blocking solution to the sample. Leave the sample on the rocker at room temperature for three hours. Then dilute the desired primary antibodies with the antibody dilution solution.Next, add 500 microliters of the antibody solution to each sample. Incubate the samples overnight at four degrees Celsius. The following day, aspirate the primary antibody solution, then wash the samples with PBST for one hour thrice at room temperature at 15 revolutions per minute.Next, dilute the appropriate secondary antibodies in five milligrams per milliliter of DAPI stock solution to achieve a final concentration of 2.5 micrograms per milliliter. Then, add 500 microliters of the solution to each sample. Next, use aluminum foil to cover the 24 well plate and incubate the plate at four degrees Celsius overnight.The next day, aspirate the secondary antibody solution and wash the samples with PBST thrice each for 30 minutes at room temperature on a rocker. Next, position a drop of hardset mounting medium on the surface of a glass slide and place the sample on the mounting medium. Finally, use nail polish to seal the samples to the glass cover slide to prevent contamination or sample movement.Here, expression of the target protein with the full length ELP under controlled conditions is validated by the presence of bands at 37 kilodaltons in both SDS-PAGE and western blot analysis. However, under uncontrolled conditions, several low molecular weight bands are also visible using western blot. The lower molecular weight bands correspond to proteins with less than four elastin-like domain repeats, indicating that the protein is not fully translated during expression.This result is common when conducting the expression above 32 degrees Celsius. To vary the cell adhesive ligand concentration while keeping the hydrogel mechanical properties constant, the ratio of ELP variant containing the fibronectin-derived cell adhesive and the non-cell adhesive sequences are tuned. Further, murine neural progenitor cells are also viable over a period of seven days when encapsulated in a 3%ELP hydrogel.The live cells are identified in green due to the calcein AM stain, whereas the red ethidium homodimer is preferentially taken up by the dead cells. To validate the stem cell phenotype of neural progenitor cells, expression of canonical markers such as Sox2 and nestin are also visualized by immunostaining when the cells are encapsulated in the 3D ELP hydrogels. After watching this video, you should have a good understanding of how to express and purify elastin-like protein for downstream encapsulation of various cell types, and in addition, investigate the role of the 3D microenvironment in modulating cellular phenotype.

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Bioengineering

12.1K Görüntüleme.  Stanford University. The overall goal of this protocol is to express and purify recombinantly derived elastin-like proteins for use as a tunable hydrogel for 3D cell encapsulation. Additionally, this protocol presents the methodology for downstream fluorescent labeling in confocal microscopy of encapsulated cells. This method can help answer key questions within the fields of biomaterials, regenerative medicine, and cell biology, such as understanding the role of cell-extracellular matrix interactions in 3D.