A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
1.3K views
⸱
July 8th, 2025
Source: Xiao, W., et al. Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells. 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.
All procedures involving sample collection have been performed in accordance with the institute'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.
Access restricted. Please log in or start a trial to view this content.
Table 1. Hydrogel formulations yielding independent control of HA concentration and mechanical properties.
| Gel Type | Part A (40 µL each) | Part B (40 µL each) | ||||
| 4Arm-PEG-MAL (50mg/mL) | Cysteine or RGD (2.81mM) | |||||
Access restricted. Please log in or start a trial to view this content.
| 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 | |
| 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 | |
| Protease/phosphatase inhibitor mini tablet | sigma aldrich | 5892970001 | |
| TrypLE express | Thermo Fisher | 12604013 | |
| 70µm cell strainer | Thermo Fisher | 22-363-548 | |
| 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 | |
| Wide orifice pipette tips | Thermo Fisher | 9405120 |
