TITLE: Generation of glioblastoma spheroid model using an orbital shaker
AUTHORS: WANNAWAT KHOTCHAWAN, Chanchao Lorthongpanich, Pakpoom Kheolamai, Sith Sathornsumetee, Surapol Issaragrisil

[DESCRIPTION]
Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor with no effective treatment but a high fatality rate. Thus, a new treatment is still urgently required. The U251 cell line is widely used as a model for investigating GBM characteristics and developing therapeutic treatments. A conventional two-dimensional (2D) culture system is routinely used for maintaining and propagating U251 cells for experiments. However, the 2D system does not correctly mimic the in vivo tumor complexity and tumor microenvironment affected GBM behaviors and responsiveness to drugs treatment. Recent research suggests that three-dimensional (3D) culture methods, such as a spheroid model, could be a better platform for mimicking tumor behavior in vivo. The 3D model can imitate cellular activity, complexity, and providing a much more accurate representation of tumor profiles and medication response in vivo. Here, we provide a simple but yet effective protocol to generate the glioblastoma spheroids from the U251 GBM cell line using an orbital shaker for investigating GBM characteristics and developing therapeutic treatments. This protocol might be applicable to other type of tumors for studying factors effecting tumor growth.

[GUIDELINES]
The size and number of spheroid model generated using an orbital shaker might vary when compared to previous techniques for spheroid generation.

[STEPS]
SECTION: Generation of three-dimensional (3D) glioblastoma spheroids
1. Culture the glioblastoma cell line (U251-MG) in the U251-MG media (DMEM-high glucose medium containing 10% fetal bovine serum) on a 75-cm2 cell culture flask for 3–4 days till the cell reach 70–80% confluence before collected for spheroid formation.
SECTION: Generation of three-dimensional (3D) glioblastoma spheroids
2. Perform cell counting using the trypan blue exclusion assay to determine cell number and viability before adjusted concentration to 1× 106 cell/ml.
SECTION: Generation of three-dimensional (3D) glioblastoma spheroids
3. Add 2 ml of culture medium (2 × 106 viable cells) into 1 well of a non-coated flat-bottom 6-well plate.

SECTION: Generation of three-dimensional (3D) glioblastoma spheroids
4. Set the orbital shaker in a CO2 incubator which previously been adjusted temperature to 37 °C and 5% CO2 atmosphere.
SECTION: Generation of three-dimensional (3D) glioblastoma spheroids
5. Place the cell culture plate in step 3 onto an orbital shaker, adjusted the speed to 120 rpm and cultured the cell  overnight.


SECTION: Generation of three-dimensional (3D) glioblastoma spheroids
6. The GBM spheres were ready to be collected for the experiment.




 






SECTION: Detection of apoptosis in glioblastoma spheroid model
7. The spheroids were collected for protein extraction followed manufacturer’s instructions.
SECTION: Detection of apoptosis in glioblastoma spheroid model
8. Briefly, lyse the spheres using cell lysis buffer for protein extraction.
SECTION: Detection of apoptosis in glioblastoma spheroid model
9. Separate the extracted protein by electrophoresis in 12% BisTris polyacrylamide gels and transferred onto PVDF membranes.
SECTION: Detection of apoptosis in glioblastoma spheroid model
10. Perform immunoblotting using primary antibodies against Caspase-3, Cleaved-caspase-3 and horseradish peroxidase conjugated secondary antibodies. Then, image the blot using an appropriate fluorescence scanner.
SECTION: Assessment of drug response in glioblastoma spheroid model
11. Culture GBM spheroids in U251-MG medium containing 0, 25, 50, and 100 μM of Temozolomide, a standard GBM drug.
SECTION: Assessment of drug response in glioblastoma spheroid model
12. Place the cell culture plate onto an orbital shaker, adjusted the speed to 120 rpm and cultured the cell for overnight in an incubator which previously been adjusted temperature to 37 °C and 5% CO2 atmosphere.
SECTION: Assessment of drug response in glioblastoma spheroid model
13. Observe spheroid morphological changes and number of dead cells after the spheroids being treated with Temozolomide for 24 hours.