Application Note 25

Human glioblastoma (U251): 3D model
demonstrates increased cell viabilityand linear drug response over 2D
Joanne Ossai1, Jonathan Sheard1,2, Darius Widera1
1
Stem Cell Biology and Regenerative Medicine Group, University of Reading
2
Sheard BioTech Ltd

INTRODUCTION

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour. Cell
culture plays an indispensable role in the cancer drug discovery process. Notably, themajority of in vitro testing of anti-cancer drugs is carried out on traditional two-dimensional(2D) cell cultures. However, 2D cell culture only poorly represents the three-dimensional(3D) tumour microenvironment. Moreover, the high stiffness of plastic and glass substrates
commonly used for 2D culture exceeds that of brain tissue resulting in stretched and unnaturalmorphology. The inability of 2D monolayers to closely approximate the pathophysiologicalconditions, has been shown to alter properties such as cell proliferation, morphology,
gene and protein expression and ultimately, drug responses. Cultivation as self-assembled
spherical clusters known as spheroids represents a widely used three-dimensional cultivation
method for glioma cells. However, due to the compact nature of these `gliomaspheres`,the diffusion of nutrients and growth factors can be compromised [1,2]. This highlights theneed for novel 3D cell culture models that could act as better disease models and moresophisticated predictors in drug development [3].
Here, we developed a novel method for the 3D cultivation of the human glioblastoma cellline U251 within the nanofibrillar cellulose hydrogel, GrowDex®. We demonstrate that 3D
cultivation of U251 cells in GrowDex results in an increased cell viability. Moreover, weshow that in contrast to 2D, U251 cells in 3D respond in a linear manner to temozolomide
which is an alkylating agent licensed for the treatment of newly diagnosed glioblastoma. This
is in line with recent reports demonstrating that three dimensional matrices can affect cancer
cell response to toxins [4].

MATERIALS• Human glioblastoma cell line U251-MG (ECACC 09063001, European Collection ofAuthenticated Cell Cultures)• Complete Media: Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 20% FBS, 1% Pen/Strep and 1% L-Glutamine (all SigmaAldrich)• GrowDex, 1.5% (Cat# 100 103 005, UPM)
• 3D culture: Low-attachment 96-well plates, (Cat# 83.3924.500,TC Plate 96 Well
Suspension F, Sarstedt)• 2D controls: Standard 96-well plates, (Cat# 83.3924.005 TC Plate 96 Well StandardF, Sarstedt)• Cell proliferation kit II XTT (Sigma Aldrich)• 4% Paraformaldehyde (PFA, Sigma Aldrich)• 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma Aldrich)• Phalloidin Atto 550 (Sigma Aldrich)• Centrifuge 5810 R, Eppendorf with a rotor suitable for 96 well tissue culture plates• Temozolomide (Sigma Aldrich)• Spectra Max 340PC384 ROM v1.21 plate reader (Molecular Devices, Wokingham,UK)• EVOS Live imaging system (ThermoFisher)• Nikon A1 plus Confocal Microscope System (Nikon Instruments Europe B.V. UnitedKingdom)

METHODS

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Figure 1. GrowDex supports 3D
cultivation of U251 glioblastoma cells.
A) U251 cultivated in 3D within 0.2%
GrowDex form homogenous networks
and display normal morphology. Scalebar: 200μm. B) XTT assay revealed that
U251 cells cultivated within 0.1, 0.2,
and 0.4% GrowDex show significantly
increased cellular viability compared tothe 2D control.
Data is displayed as mean of three
independent experiments ± SEM. ns:
not significant. ****P <0.001.

U251 cells in 2D and 3D were cultured in complete media and incubated in ahumidified incubator at 37°C and 10% CO2.
For 2D culture, cells were resuspended in complete media at a concentration of 1000
cells/μL and cultivated in 96 well cell culture plates.
For 3D culture, cells were mixed with complete media and GrowDex to provide a final
concentration of 100 μL of 0.1, 0.2 or 0.4% GrowDex seeded with 1000 cells/µl.
Embedded cells were transferred into 96 well tissue culture plates and fed with 50 µl
complete media. Cells were maintained in a humidified incubator at 37°C and 10%
CO2 and the medium was replaced every 2 to 3 days.
For the viability assessment, 50 μL the of XTT labelling mixture, was added to each wellof the 96-well plate and the plates were incubated for 4 h in a humidified incubator at37°C, 10% CO2 followed by measurement of the absorbance of the XTT formazan
metabolite.

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7.

DNA/Actin

BDNA/Actin

3D reconstruction

AFor the phalloidin/DAPI staining, the culture media was carefully removed from cells
in 96-well plate. Cell cultures were fixed with 4% (w/v) paraformaldehyde solution inphosphate-buffered saline (PBS) for 20 min. The fixative solution was removed, andcells were washed with PBS. Cells were permeabilized with 0.1% (v/v) TritonX-100 in
PBS for 30 min at room temperature. To remove the permeabilization solution, the plate
was centrifuged at 100g for 30 sec. Lyophilisate of phalloidin (10 mnol) was dissolvedin 500 µl of methanol. The cells were stained overnight at 4°C using 30 µL of thephalloidin stock diluted in 1 ml PBS containing DAPI (1:1500).
Phase contrast images were taken using the EVOS imaging system whereas thephalloidin and DAPI stained cells were imaged on the Nikon A1 plus Confocal
Microscope System.
U251 cells cultivated in 2D and 3D were exposed to increasing concentrations oftemozolomide at 37°C at 10% CO2 for 48 h and subjected to XTT-based viability
assessment.

Figure 2. U251 cells are evenly distributed in all three dimensions of the GrowDex. Cells
were cultivated in 0.2% GrowDex and stained for actin with Atto-coupled phalloidin.
Nuclear counterstaining was performed with DAPI. A) Actin staining and confocal laser
scanning microscopy revealed an isotropic distribution of cells in all three dimensions of theGrowDex hydrogel. Scale bar: 100 μm. B) 3D reconstruction.

RESULTS

Phase contrast microscopy and XTT viability assays revealed that U251 grow homogenously
within GrowDex and that 3D cultivation results in a significantly increased cellular viability
compared to 2D controls (Fig. 1). Moreover, we show using confocal laser scanningmicroscopy that U251 cells are evenly distributed in all three dimensions of the hydrogel (Fig.
2). Finally, a drug response assay showed that 3D cultivation leads to a linear reduction ofviability in response to the cytotoxic temozolomide stimulus (Fig. 3).

Figure 3. 3D cultivation within GrowDex results in a linear response to the cytotoxic
drug temozolomide. U251 cells cultivated in 2D and 3D were exposed to increasing
concentrations of temozolomide followed by assessment of viability using XTT. Statisticalevaluation of the data revealed that cells within 3D respond to the toxic stimulus in a linear
manner whereas only high concentration of temozolomide significantly reduce the viability
when cultured in 2D. Data is displayed as mean of three independent experiments ± SEM.
ns: not significant. ****P < 0.001.

CONCLUSIONSREFERENCES

Our results show that GrowDex is a suitable scaffold for 3D cultivation of the humanglioblastoma cell line U251. Moreover, we show that GrowDex leads to an increased
viability over concentration range from 0.1-0.4% when compared to 2D culture. Finally,
we demonstrate that 3D culture in GrowDex can mediate a more linear drug response incomparison to conventional 2D culture. The results indicate that 3D cultivation of glioma cells
within GrowDex might better reflect the pathophysiological environment and could improvecurrent 2D cell-based drug screening platforms. Notably, our method could be easily
adopted for primary glial cells, other glioma cell lines, and primary glioma cell cultures.

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UPM Biomedicals
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Edmondson, R., et al. (2014). "Three-Dimensional Cell Culture Systems and Their
Applications in Drug Discovery and Cell-Based Biosensors." Assay and Drug
Development Technologies 12(4): 207-218.
Laks, D. R., et al. (2016). "Large-scale assessment of the gliomasphere model system."
Neuro Oncol 18(10): 1367-1378.
Matak, D., et al. (2017). "Colony, hanging drop, and methylcellulose three dimensional
hypoxic growth optimization of renal cell carcinoma cell lines." Cytotechnology 69(4):565-578.
Zustiak, S. P., et al. (2016). "Three-dimensional matrix stiffness and adhesive ligands
affect cancer cell response to toxins." Biotechnol Bioeng 113(2): 443-452.

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