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.
Protocol
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. 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 …
Representative Results
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)</…
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