Published: August 24th, 2018
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....
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.
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.......
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.......
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....
|Any pH meter that has pH 2-10 sensitivity
|General lab equipment
|Erlenmeyer flask (125mL)
|2L polypropylene beaker
|500-750 kDa range
|1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
|Hydrochloric acid (HCl)
|Sodium hydroxide (NaOH)
|Ellman's test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid
|Deuterated water (deuterium oxide)
|0.22µm vacuum driven filter
|Phosphate buffered saline (PBS)
|Hanks' balanced salt saline (HBSS)
|molecular weight around 20kDa
|molecular weight around 20kDa
|RGD ECM mimetic peptide
|Custom peptide with sequence "GCGYGRGDSPG", N-terminal should be acetylated
|Use razor blade to cut into single pieces
|complete culture medium
|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
|protease/phosphatase inhibitor mini tablet
|70µm cell strainer
|Dissolve 4% (w/v) in PBS, keep pH 7.4
|Optimal Cutting Temperature (O.C.T.) compound
|Cell culture incubator
|Any General One with 5% CO2 and 37C
|Any General Lab equipment with -20C and -80C capacity
|Disposable embedding molds
|Any -105C freeze dryers
|Wide orifice pipette tips
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