Name: hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells
Uploaded: 2018-08-24T14:30:00
Description: Watch this Scientific Journal Video about Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells at JoVE.com

This work was supported with funding from the NIH (R21NS093199) and the UCLA ARC 3R's Award. Our sincerest thanks go to the lab of Dr. Harley Kornblum for provision of the HK301 and HK157 cell lines. We also thank UCLA Tissue Pathology Core Laboratory (TPCL) for cryosectioning, Advanced Light Microscopy/Spectroscopy core facility (ALMS) in California Nanosystems Institute (CNSI) at UCLA for use of the confocal microscope, UCLA Crump Institute for Molecular Imaging for using IVIS imaging system, UCLA Molecular Instrumentation Center (MIC) for providing magnetic resonance spectroscopy, and Flow Cytometry Core in Jonsson Comprehensive Cancer Center (JCCC) at UCLA for providing instrumentation for flow cytometry.

All human tissue collection steps were carried out under institutionally approved protocols.
1. Thiolation of Hyaluronic Acid
Note: Molar ratios are stated with respect to 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 ...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+orthotopic+glioblastoma+xenograft+models+recapitulate+the+histopathology+and+biology+of+human+glioblastomas+in+situ.">Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ.</a> Cell Reports. 3 (1), 260-273 (2013).</li><li>Oh, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+validation+of+personalized+treatment+strategy+based+on+genetic+characteristics+of+glioblastoma.">Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma.</a> PloS one. 9 (8), e103327 (2014).</li><li>Pedron, S., Becka, E., Harley, B. A. 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Generation of reproducible data using this 3D culture system requires: 1) consistent batch-to-batch thiolation of HA, 2) practice to achieve efficient mixing of hydrogel precursors and handling of hydrogel cultures to prevent damage and 3) optimized seeding density for each cell line used.
When a particular weight percentage of HA is desired in the hydrogel, the degree of thiolation of HA determines the crosslink density. We recommend using a consistent amount of HA for each thiolation reactio...

Three-dimensional (3D) culture systems recapitulate interactions between cells and their surrounding extracellular matrix (ECM) in native tissues better than their two-dimensional (2D) counterparts1,2. Advancements in tissue engineering have yielded sophisticated, 3D culture platforms that enable controlled investigations into 1) how chemical and physical components of the matrix microenvironment affect cell behaviors and 2) efficacy of new therapeutic strategies for a number of diseases, including cancers2. While in vitro models cannot account for systemic factors, such as endocrine and immune signals, and thus cannot completely replace in vivo models, they provide several advantages including reproducibility, experimental control, affordability and speed. Here, we describe the use of brain-mimetic hydrogels in which 3D cultures of patient-derived brain tumor cells capture many aspects of tumor physiology, in particular, the dynamics of acquiring treatment resistance3. Compared to other in vitro methods, these cultures better represent in vivo tumor models and clinical observations3.
Glioblastoma (GBM) is the most frequent and lethal cancer originating in the brain, with a median survival of only 1-2 years4,5. In recent years, many studies have focused on the influence of tumor matrix environment in GBM6,7,8. The unique brain ECM has been reported to affect GBM cell migration, proliferation, and therapeutic resistance6,7,8,9,10,11,12. Hyaluronic acid (HA) is an abundant glycosaminoglycan (GAG) in the brain, where it interacts with other GAGs and proteoglycans to form a hydrogel-like mesh13. Many studies have reported HA overexpression in GBM tumors and its subsequent effects on cancer progression8,9,13,14,15,16,17. Other ECM components also affect GBM tumor growth and invasion6,7,15,18. For example, fibronectin and vitronectin, which are typically overexpressed in GBM, induce heterodimerization of cell surface integrin receptors through binding to the &#34;RGD&#34; sequence and initiate complex signaling cascades that promote tumor survival19,20,21. Besides biochemical influences, physical properties of the tissue matrix also affect GBM progression22,23.
Continual acquisition of resistance to therapies is one of the main drivers of GBM lethality4. Drugs showing promising results in 2D or gliomasphere models have failed in subsequent animal studies and clinical cases3, indicating that the effects of microenvironmental factors significantly contributed to GBM tumor response1. While animal models can provide a 3D, physiologically appropriate microenvironment to xenografted patient cells and generate clinically relevant outcomes24,25, the complexity of the brain microenvironment in vivo makes it challenging to determine which features, including cell-matrix interactions, are key to specific biological outcomes. Identification of new therapeutic targets will benefit from the use of simplified culture platforms in which biochemical and biophysical properties are defined.
Unlike previously reported biomaterial models of the GBM tumor microenvironment26,27 which have not achieved true orthogonal control over individual biochemical and physical features of the ECM, the biomaterial platform reported here enables decoupling of the contributions of multiple independent features to GBM cell phenotype. Here, we present an HA-based, orthogonally tunable, hydrogel system for 3D culture of patient-derived GBM cells. Hydrogels are formed from two polymer components: 1) biologically active HA and 2) biologically inert polyethylene glycol (PEG). PEG is a widely used biocompatible and hydrophilic material with low protein adsorption and minimal immunogenicity28. Here, approximately 5% of glucuronic acid moieties on HA chains are functionalized with thiol groups to enable crosslinking to a commercially available 4-arm-PEG terminated with maleimides via Michael-type addition. In its most common form in the body, HA exists in high molecular weight (HMW) chains. Here, a low degree of modification of HMW HA (500-750 kDa) helps to preserve native interactions of HA and its cell receptors, including CD4429. By substituting PEG-thiol for HA-thiol while maintaining a constant molar ratio of total thiols to maleimides, HA concentration can be decoupled from mechanical properties of the resulting hydrogels. Furthermore, stoichiometric controls can be used to conjugate cysteine-terminated peptides to a defined average number of maleimide-terminated arms on each 4-arm-PEG. Incorporation of ECM-derived, adhesive peptides enables interactions with integrins on cultured cells, through which biochemical and chemical signals are transduced1. Maleimide-thiol addition occurs very quickly under physiological conditions, minimizing the time required for cell encapsulation and maximizing survival of patient-derived cells. Moreover, hydrogel cultures can be treated like typical tissue specimen and are compatible with standard characterization techniques including Western blotting, flow cytometry, and immunofluorescence staining. The following protocol describes the procedures for fabricating hydrogels, establishing 3D cultures of patient-derived GBM cells and techniques for biochemical analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:2204px;" width="2203">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">pH meter</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td style="width:1579px;">Any pH meter that has pH 2-10 sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Stir plate</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>General lab equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Erlenmeyer flask (125mL)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">FB-501-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">dialysis tubes</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">21-152-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">2L polypropylene beaker</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">S01916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">sodium hyaluronan</td>
			<td style="width:136px;">Lifecore</td>
			<td style="width:121px;">HA700k-5</td>
			<td>500-750 kDa range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">PI-22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">N-hydroxysuccinimide (NHS)</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">130672-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Hydrochloric acid (HCl)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">SA48-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Sodium hydroxide (NaOH)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">SS266-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Cystamine dihydrochloride</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC111770250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dithiolthreitol (DTT)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP172-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Ellman&#39;s test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:121px;">D218200-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Deuterated water (deuterium oxide)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC166301000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.22&#181;m vacuum driven filter</td>
			<td style="width:136px;">CellTreat</td>
			<td style="width:121px;">229706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Phosphate buffered saline (PBS)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">P32080-100T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Hanks&#39; balanced salt saline (HBSS)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">MT-21-022-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">4-arm-PEG-maleimide</td>
			<td style="width:136px;">JenKem Technology</td>
			<td style="width:121px;">A7029-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">4-arm-PEG-thiol</td>
			<td style="width:136px;">JenKem Technology</td>
			<td style="width:121px;">A7039-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">L-Cysteine&#160;</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">C7880-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">RGD ECM mimetic peptide</td>
			<td style="width:136px;">Genscript Biotech</td>
			<td style="width:121px;">N/A</td>
			<td>Custom peptide with sequence &#34;GCGYGRGDSPG&#34;, N-terminal should be acetylated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">silicone molds</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:121px;">GBL664201-25EA</td>
			<td>Use razor blade to cut into single pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">complete culture medium</td>
			<td style="width:136px;">Various</td>
			<td style="width:121px;">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 height="21">
			<td height="21" style="height:21px;width:368px;">patient derived GBM cell</td>
			<td style="width:136px;">N/A</td>
			<td style="width:121px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">20G needle</td>
			<td style="width:136px;">BD medical</td>
			<td style="width:121px;">305175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">1mL syringe</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">14-823-434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">10mL syringe</td>
			<td style="width:136px;">BD medical</td>
			<td style="width:121px;">302995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">RIPA Buffer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">PI-89901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">protease/phosphatase inhibitor mini tablet</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">5892970001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">vortex shaker</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12-814-5Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">TrypLE express</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">70&#181;m cell strainer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Paraformaldehyde</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC416785000</td>
			<td>Dissolve 4% (w/v) in PBS, keep pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">D-sucrose</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP220-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Optimal Cutting Temperature (O.C.T.) compound</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">NC9373881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Cell culture incubator</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>Any General One with 5% CO2 and 37C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">fridge/freezer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>Any General Lab equipment with -20C and -80C capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Disposable embedding molds</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lyapholizer</td>
			<td style="width:136px;">Labconco</td>
			<td style="width:121px;">N/A</td>
			<td>Any -105C freeze dryers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">HEPES</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Amber vial</td>
			<td style="width:136px;">Kimble Chase</td>
			<td style="width:121px;">60912D-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Wide orifice pipette tips</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">9405120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">2-methylbutane</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">03551-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dry Ice</td>
			<td style="width:136px;">N/A</td>
			<td style="width:121px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

For each batch of thiolated HA, the degree of thiolation should be verified using H1-NMR or an Ellman&#39;s test. HA modification using the procedure described here consistently generates ~5% thiolation (defined as the molar ratio of thiols to HA disaccharides) (Figure 1).
Setting up this new culture platform will require each laboratory to perform rigorous testing to ens...

Watch this Scientific Journal Video about Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells at JoVE.com

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

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

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...