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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Bioengineering

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

Published: August 24th, 2018

DOI:

10.3791/58176

1Department of Bioengineering, University of California, Los Angeles, 2Department of Bioengineering, Jonsson Comprehensive Cancer Center, Broad Stem Cell Research Center, Brain Research Institute, University of California, Los Angeles

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 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.

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 end....

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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.

  1. 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 .......

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For each batch of thiolated HA, the degree of thiolation should be verified using H1-NMR or an Ellman'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.......

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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.......

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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 pro....

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Name Company Catalog Number Comments
pH meter Thermo Fisher N/A Any pH meter that has pH 2-10 sensitivity
Stir plate Thermo Fisher N/A General lab equipment
Erlenmeyer flask (125mL) Thermo Fisher FB-501-125
dialysis tubes Thermo Fisher 21-152-14
2L polypropylene beaker Thermo Fisher S01916
sodium hyaluronan Lifecore HA700k-5 500-750 kDa range
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) Thermo Fisher PI-22980
N-hydroxysuccinimide (NHS) sigma aldrich 130672-5G
Hydrochloric acid (HCl) Thermo Fisher SA48-500
Sodium hydroxide (NaOH) Thermo Fisher SS266-1
Cystamine dihydrochloride Thermo Fisher AC111770250
Dithiolthreitol (DTT) Thermo Fisher BP172-25
Ellman's test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid Sigma Aldrich D218200-1G
Deuterated water (deuterium oxide) Thermo Fisher AC166301000
0.22µm vacuum driven filter CellTreat 229706
Phosphate buffered saline (PBS) Thermo Fisher P32080-100T
Hanks' balanced salt saline (HBSS) Thermo Fisher MT-21-022-CV
4-arm-PEG-maleimide JenKem Technology A7029-1 molecular weight around 20kDa
4-arm-PEG-thiol JenKem Technology A7039-1 molecular weight around 20kDa
L-Cysteine  sigma aldrich C7880-100G
RGD ECM mimetic peptide Genscript Biotech N/A Custom peptide with sequence "GCGYGRGDSPG", N-terminal should be acetylated
silicone molds Sigma Aldrich GBL664201-25EA Use razor blade to cut into single pieces
complete culture medium Various Various 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 25µg/mL (Sigma Aldrich), culture medium varies in different labs
patient derived GBM cell N/A N/A
20G needle BD medical 305175
1mL syringe Thermo Fisher 14-823-434
10mL syringe BD medical 302995
RIPA Buffer Thermo Fisher PI-89901
protease/phosphatase inhibitor mini tablet sigma aldrich 5892970001
vortex shaker Thermo Fisher 12-814-5Q
TrypLE express Thermo Fisher 12604013
70µm cell strainer Thermo Fisher 22-363-548
Paraformaldehyde Thermo Fisher AC416785000 Dissolve 4% (w/v) in PBS, keep pH 7.4
D-sucrose Thermo Fisher BP220-1
Optimal Cutting Temperature (O.C.T.) compound Thermo Fisher NC9373881
Cell culture incubator Thermo Fisher N/A Any General One with 5% CO2 and 37C
fridge/freezer Thermo Fisher N/A Any General Lab equipment with -20C and -80C capacity
Disposable embedding molds Thermo Fisher 12-20
Lyapholizer Labconco N/A Any -105C freeze dryers
HEPES Thermo Fisher BP310-500
Amber vial Kimble Chase 60912D-2
Wide orifice pipette tips Thermo Fisher 9405120
2-methylbutane Thermo Fisher 03551-4
Dry Ice N/A N/A

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