eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Thiolation of Hyaluronic Acid
NOTE: Molar ratios are stated with respect to a total number of carboxylate groups unless otherwise specified.
<ol>
	<li>Dissolve 500 mg of sodium hyaluronate (HA, 500-750 kDa) at 10 mg/mL in deionized, distilled water (DiH2O) in an autoclave sterilized 250 mL Erlenmeyer flask. Stir the solution (~200 rpm) at room temperature for 2 hours to fully dissolve HA. Use a stir bar and magnetic stir plate to keep the reaction stirring during the thiolation procedure.</li>
	<li>Using 0.1 M hydrochloric acid (HCl), adjust the pH of the HA solution to 5.5. Weigh out 69.6 mg (0.25x molar ratio) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). 
	NOTE: Although undissolved EDC can be added directly to the HA solution, it is typically easier to dissolve EDC first in 1 mL of DiH2O and then quickly add the EDC solution to the HA solution. Pre-dissolution in 1 mL of DiH2O can also be done with N-hydroxysuccinimide (NHS) and cystamine dihydrocholoride before adding to the reaction in steps 1.3 and 1.4.</li>
	<li>Add 17.9 mg (0.125x molar ratio) of NHS to the reaction. Adjust the pH of the reaction solution to 5.5 using 0.1 M HCl and incubate the reaction at room temperature for 45 minutes.</li>
	<li>Add 70.0 mg (0.25x molar ratio) of cystamine dihydrochloride into the reaction solution. Use 0.1 M sodium hydroxide (NaOH) to adjust pH to 6.25. Incubate the reaction at room temperature overnight while stirring.</li>
	<li>On the following day, use 1 M NaOH to adjust the pH of the reaction solution to around 8. Add 192 mg (4x molar ratio) of dithiothreitol (DTT) to the reaction. Adjust the pH back to 8 after the addition of DTT and leave stirring for 1-2 hours at room temperature.</li>
	<li>Use 1 M HCl to adjust pH to 4. Transfer the entire reaction mixture into a pre-soaked dialysis membrane (molecular weight cut-off around 13 kDa) and dialyze against DiH2O pre-adjusted to pH 4. 
	NOTE: The volume of DiH2O for dialysis should be at least 40 times the volume of the reacted HA solution (e.g., 50 mL sample in 2 L of DiH2O, pH 4).</li>
	<li>Replace the dialysis solution (DiH2O, pH 4) twice daily for 3 days at room temperature.</li>
	<li>Filter the dialyzed solution through a 0.22 &#181;m membrane, vacuum-driven filter. Flash freeze solution of thiolated HA in liquid nitrogen. Use a lyophilizer to freeze dry samples over 2 days. Thiolated HA in dry form can be stored in a vacuum-sealed desiccator at -20 &#176;C for at least 6 months.</li>
	<li>To determine the degree of thiolation, use Ellman&#39;s test and/or proton NMR spectroscopy. 
	&#160;</li>
</ol>
2. Preparation of Crosslinking Materials
<ol>
	<li>Dissolve thiolated HA at the desired concentration (in this example, 13.3 mg/mL) in HEPES-buffered saline (20 mM HEPES in Hank&#39;s balanced salt saline (HBSS) buffer, adjusted to fall within a pH of 8-9) in an amber vial to minimize exposure to ambient light. Keep the solution stirring constantly. 
	NOTE: Formation of disulfide bonds between thiols conjugated to HA may occur if the pH is above 8, the solution concentration is too high, or the solution is left stirring for too long before gelation. To avoid this issue, use below 15 mg/mL of thiolated HA and dissolve thiolated HA within 2 hours of forming hydrogels.</li>
	<li>Dissolve 4-arm-PEG-maleimide (PEG-Mal, 20 kDa) and 4-arm-PEG-thiol (PEG-thiol, 20 kDa) in phosphate buffered saline (PBS), pH 7.4, to prepare 50 mg/mL PEG-Mal and PEG-thiol stock solutions. 
	NOTE: It takes at least 1 hour to fully dissolve PEG reagents.</li>
	<li>If adding ECM-derived peptides, dissolve cysteine-containing peptides in PBS to prepare a stock solution. 
	NOTE: Use a stock concentration of 2-4 mM.</li>
	<li>Soak silicone rubber molds in ethanol for at least 20 min to clean them and then autoclave them for sterilization.</li>
</ol>
3. Hydrogel Crosslinking and Cell Encapsulation
NOTE: As an example here, the encapsulation of four individual, 80 &#181;L hydrogels with 0.5% (w/v) HA and compressive modulus of 1 kPa is described3. Please see Table 1, for example, recipes that yield hydrogels with varying properties: two hydrogels incorporating the integrin-binding peptide RGD and two hydrogels incorporating cysteine caps as a negative control for peptide activity. The seeding concentration of patient-derived GBM cells is 500,000 cells/mL.
<ol>
	<li>Dilute the PEG-Mal stock solution (50 mg/mL, from step 2.2) to 12.5 mg/mL by adding 40 &#181;L of the stock solution to 120 &#181;L PBS. Split the diluted solution into two 1.5 mL microcentrifuge tubes, each with 80 &#181;L.</li>
	<li>Add 16 &#181;L of stock RGD solution (2.8 mM, from step 2.3) to one tube and 16 &#181;L of stock cysteine solution (2.8 mM, from step 2.3) to the second tube. Vortex to mix. Place the PEG-Mal-RGD or PEG-Mal-CYS solutions on ice until use in step 3.9. 
	NOTE: The procedure as described here yields hydrogels with ~140 &#181;M of peptide. This is equivalent to approximately 1 out of every 8 available PEG arms being occupied with a peptide. This can be varied by altering the molar ratio of cysteine-terminated peptides to available maleimide groups. In general, a maximum concentration of 280 &#181;M of peptide can be achieved while still leaving sufficient numbers of maleimide groups available for hydrogel crosslinking.</li>
	<li>Dilute the PEG-thiol stock solution (50 mg/mL, from step 2.2) to 5 mg/mL by mixing 4 &#181;L of 50 mg/mL stock solution with 36 &#181;L of PBS. Add 120 &#181;L of dissolved, thiolated HA from step 2.1 to the mixture. 
	NOTE: Solutions of HMW HA are very viscous. Thus, we recommend using a wide-orifice micropipette tip to transfer solutions. Pipette slowly to avoid the solution sticking to the walls of the micropipette tip and to improve measurement accuracy.</li>
	<li>Place the clean, dry molds (prepared in step 2.4) into each well of a non-tissue culture-treated 12-well plate. Using the clean, blunt end of a pipette tip, press the gel mold and double-check the sealing between the molds and the bottom of the well plate. 
	NOTE: Check the seal again right before encapsulation to prevent leakage.</li>
	<li>Passage cultured GBM cells, dissociate to single cells and determine cell concentration as previously described3. 
	NOTE: Some GBM lines cannot be dissociated into single cells. In this case, whole gliomaspheres can be encapsulated. However, prepare dissociated single cells after precise cell counting to improve reproducibility. The protocol for gliomasphere passaging varies among different labs and many of these are likely compatible with hydrogel encapsulation.
	<ol>
		<li>(Recommended) Centrifuge gliomaspheres at 500 x g for 5 min. Remove the supernatant and add 1 mL of cell dissociation enzyme to the cell pellet. Then, incubate for 5 min, gently tapping the tube to agitate.</li>
		<li>Add 4 mL of complete culture medium (50 ng/mL EGF, 20 ng/mL FGF-2, 25 &#181;g/mL heparin, G21 supplement, and 1% penicillin/streptomycin in DMEM/F12) to the cells.</li>
		<li>Centrifuge again at 500 x g for 5 min and remove the supernatant. Finally, resuspend the cell pellet in 1 mL of the complete medium and pass suspended cells through a 70 &#181;m cell strainer. (Recommended) wash the strainer with an additional 4 mL of the complete medium to maximize the number of cells recovered.</li>
	</ol>
	</li>
	<li>Estimate the concentration of cells in the suspension using a hemocytometer. Split the cell suspension into 2 centrifuge tubes, where each tube contains ~80,000 cells. Centrifuge at 500 x g for 5 min; generally, 80,000 cells will make up 2 80 &#181;L hydrogels.</li>
	<li>Remove the supernatant and resuspend one pellet in 80 &#181;L of PEG-MAL-RGD solution and a second pellet in 80 &#181;L of PEG-MAL-CYS solution (prepared in step 3.1).</li>
	<li>Using a 200 &#181;L, wide-orifice micropipette tip, dispense 40 &#181;L of HA solution (from step 3.3 above) into each rubber silicone mold (as prepared in step 3.4).</li>
	<li>Using a 200 &#181;L, wide-orifice micropipette tip, mix the 40 &#181;L of PEG-MAL-CYS or PEG-MAL-RGD cell solution with the HA solution in the mold. Pipette up and down quickly no more than 10 times. Repeat for each gel culture being prepared. 
	NOTE: This step takes practice, since initial gelation occurs quickly (within 30 s). Pipette up and down while moving the tip to different locations in molds to ensure even mixing. Always keep the tip below liquid level to avoid formation of air bubbles. Do not mix the solution too many times as gel may get formed inside the micropipette tip. PEG-MAL-CYS and PEG-MAL-RGD can be combined at varying ratios to achieve the desired RGD peptide concentration.</li>
	<li>Place the well-plate containing gel-encapsulated cells into a 37 &#176;C cell culture incubator for 5-10 minutes to ensure completion of the reaction.</li>
	<li>Add 2-2.5 mL of culture medium to each well with formed gels. Use a sterile, 2 &#181;L pipette tip or micro-spatula, to gently separate the mold and gel. Use pre-sterilized forceps to remove the mold from the well plate. Place the well plate back to cell incubator (37 &#176;C and 5%CO2) for culture and future experiments. 
	NOTE: Pull the mold vertically upward to avoid harming the gel cultures. In general, medium in gel cultures should be replaced every 3-4 days. Take care not to aspirate hydrogels when removing the medium. If this is an issue, we recommend using a plastic transfer pipette. If bioluminescence imaging to track cell number is planned, GBM cells must be transduced with lentivirus encoding constitutively luciferase expression before encapsulation, as previously described3. For bioluminescence imaging, add 1 mM of D-luciferin to the culture medium 1 h prior to luminescence imaging.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>pH meter</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Any pH meter that has pH 2-10 sensitivity</td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>General lab equipment</td>
		</tr>
		<tr>
			<td>Erlenmeyer flask (125mL)</td>
			<td>Thermo Fisher</td>
			<td>FB-501-125</td>
			<td></td>
		</tr>
		<tr>
			<td>2L polypropylene beaker</td>
			<td>Thermo Fisher</td>
			<td>S01916</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hyaluronan</td>
			<td>Lifecore</td>
			<td>HA700k-5</td>
			<td>500-750 kDa range</td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Thermo Fisher</td>
			<td>PI-22980</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>sigma aldrich</td>
			<td>130672-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Thermo Fisher</td>
			<td>SA48-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Thermo Fisher</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cystamine dihydrochloride</td>
			<td>Thermo Fisher</td>
			<td>AC111770250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiolthreitol (DTT)</td>
			<td>Thermo Fisher</td>
			<td>BP172-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ellman&#39;s test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid</td>
			<td>Sigma Aldrich</td>
			<td>D218200-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterated water (deuterium oxide)</td>
			<td>Thermo Fisher</td>
			<td>AC166301000</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22&#181;m vacuum driven filter</td>
			<td>CellTreat</td>
			<td>229706</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Thermo Fisher</td>
			<td>P32080-100T</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt saline (HBSS)</td>
			<td>Thermo Fisher</td>
			<td>MT-21-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>4-arm-PEG-maleimide</td>
			<td>JenKem Technology</td>
			<td>A7029-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr>
			<td>4-arm-PEG-thiol</td>
			<td>JenKem Technology</td>
			<td>A7039-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>sigma aldrich</td>
			<td>C7880-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>RGD ECM mimetic peptide</td>
			<td>Genscript Biotech</td>
			<td>N/A</td>
			<td>Custom peptide with sequence &#34;GCGYGRGDSPG&#34;, N-terminal should be acetylated</td>
		</tr>
		<tr>
			<td>Silicone molds</td>
			<td>Sigma Aldrich</td>
			<td>GBL664201-25EA</td>
			<td>Use razor blade to cut into single pieces</td>
		</tr>
		<tr>
			<td>Complete culture medium</td>
			<td>Various</td>
			<td>Various</td>
			<td>DMEM/F12 (Thermofisher) with non-serum supplement (G21 from GeminiBio), epidermal growth factor 50ng/mL (Peprotech), fibroblast growth factor 20ng/mL (Pepro Tech) and heprain 25&#181;g/mL (Sigma Aldrich), culture medium varies in different labs</td>
		</tr>
		<tr>
			<td>Patient derived GBM cell</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease/phosphatase inhibitor mini tablet</td>
			<td>sigma aldrich</td>
			<td>5892970001</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Thermo Fisher</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>70&#181;m cell strainer</td>
			<td>Thermo Fisher</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Any General One with 5% CO2 and 37C</td>
		</tr>
		<tr>
			<td>Fridge/freezer</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Any General Lab equipment with -20C and -80C capacity</td>
		</tr>
		<tr>
			<td>Disposable embedding molds</td>
			<td>Thermo Fisher</td>
			<td>12-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide orifice pipette tips</td>
			<td>Thermo Fisher</td>
			<td>9405120</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hyaluronic acid-based hydrogel for 3-dimensional culture of glioblastoma cells

Source: Xiao, W.,&#160;et al. <a href="https://app.jove.com/v/58176">Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells.</a> J. Vis. Exp. (2018).
This video demonstrates how gliomaspheres, derived from brain tumors, are separated into single cells and encapsulated in a hyaluronic acid-based hydrogel. This process promotes cell-cell interactions, forming a structured, three-dimensional glioblastoma model.

Table 1. Hydrogel formulations yielding independent control of HA concentration and mechanical properties.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Gel Type </td>
			<td colspan="3">Part A (40 &#181;L each)</td>
			<td colspan="3">Part B (40 &#181;L each)</td>
		</tr>
		<tr>
			<td>4Arm-PEG-MAL (50mg/mL)</td>
			<td>Cysteine or RGD (2.81mM)</...

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Thiolation of Hyaluronic Acid
...

Begin with gliomaspheres, cell aggregates derived from a brain tumor or glioblastoma.
Add dissociation enzymes. Incubate to separate the glioblastoma cells and obtain a single-cell suspension.
Next, introduce a serum-enriched culture medium to stop the enzyme activity.
Centrifuge and remove the supernatant, then resuspend the cells.
Filter the suspension, then centrifuge and remove the supernatant.
Resuspend the cells in hydrogel precursors that contain small peptides.
Load this mixture into wells with a pre-assembled silicone mold containing hyaluronic acid.
Mix the solution. Incubate to allow the interaction between the hyaluronic acid and hydrogel precursors, forming a hydrogel that facilitates cell entrapment.
Overlay the hydrogel with culture media to sustain cell survival.
After disassembling the mold, incubate the hydrogel.
Within the hydrogel, the adhesion proteins on cells interact with the precursor peptides and hyaluronic acid.
This causes cells to form small clusters and develop a three-dimensional culture.

a-hyaluronic-acid-based-hydrogel-for-3-dimensional-culture

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

118 ビュー. 出典:Xiao, W., et al. 患者由来の膠芽腫細胞の3次元培養のためのヒアルロン酸ベースのハイドロゲル。J. Vis. Exp. (2018年)。 このビデオは、脳腫瘍に由来する神経膠腫球が単一細胞に分離され、ヒアルロン酸ベースのハイドロゲルにカプセル化される様子を示しています。このプロセスにより、細胞間相互作用が促進され、構造化された三次元神経膠芽腫モデルが形成されます?...

ビデオ: 膠芽腫細胞の3次元培養のためのヒアルロン酸ベースのハイドロゲル - 概念

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

神経膠芽腫の3次元スフェロイドモデル

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

JoVE Journal

がん研究

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

生物工学

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.

ライブ顕微鏡イメージング下での3Dヒドロゲルの制御株

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

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

記録

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells