Application Note 31

Solid tumor derived cell line BT474
and ascites metastasis derived cellline COLO205 cultures in GrowDex®
Juha Rantala1, Lauri Paasonen2
1
Misvik Oy, Finland
2
UPM Biomedicals, Finland

INTRODUCTION

Better in vitro cancer cell models are needed for e.g. cancer biology research and drug
development. Traditional 2D cell cultures are being replaced with more functional andmore physiologically relevant 3D cell models. Cancer cells derived from solid tumors orliquid biopsies, such pleural or ascites effusion, may require different culturing conditions.
Anchorage-independency is a cellular feature facilitating the cells’ capacity to divide andfunction in the absence of surface to anchor with. Solid tissue cells may enter a programmed
cell death (anoikis) when detached from their physical extracellular matrix (ECM), whereas
metastatic cancer cells may bypass this mechanism in order to survive outside the primarytumor e.g. in bloodstream or effusions (1). Previously it has been demonstrated that
nanofibrillar cellulose hydrogel, GrowDex®, offers a suitable 3D culture matrix for variouscell types (2, 3, 4).
In this study we investigated the culture of two cancer cell lines in three conditions: 2D
monolayer, in suspension, and in 3D using GrowDex. BT474 is a breast ductal carcinoma
cell line derived from solid tumor, and COLO205 is a colorectal adenocarcinoma cell line
derived from ascites metastasis.

MATERIALS• GrowDex® 1.5% (Cat No. 100103005, UPM)
• Cell culture medium• RPMI-1640 medium (Cat No. BE12-7627, Lonza)• 2mM L-Glutamine (Cat No. 25030, Gibco)• 1% Penicillin-Streptomycin (Cat No. 15140, Gibco)• 10% Fetal Bovine Serum (Cat No. S181B-500, Biowest)• Poly(2-hydroxyethyl methacrylate, pHEMA (Cat No. 18894, Polysciences Inc.)• CellTiter-Glo (Cat No. 8462, Promega).

METHODS• To minimize 2D cell growth on the well bottom, microwells for suspension and GrowDex
cultures were coated with 1.2% pHEMA .
• For coating pHEMA was mixed with ethanol and 40 µl pipetted into the microwells. The
coating was ready when all alcohol had evaporated leaving a thin hydrophobic film
covering the plastic surface.
• Wells for conventional 2D monolayer culture were left untreated.
• Cultures in GrowDex were performed with 0.4% GrowDex concentration.
• Cell seeding density was 8000 cells / well in 100 µl with 50 µl medium on top.
• Cells were incubated in 5% CO2 , +37°C and the medium was refreshed every two or
three days.
• Cell growth was monitored daily by microscopy using Motic AE2000 inverted
microscope. Images were acquired on days 1, 3 and 6 using the 20x objective.
• Cell viability was quantified on days 1 and 6 using CellTiter-Glo® luminescent assay,
whereby 50 μl assay reagent was pipetted directly into wells and samples incubatedat +37°C for 1 h. Luminescence was quantified using the Labrox plate reader (Labrox,Finland).

RESULTS AND DISCUSSION

BT474 cells formed loose clusters in suspension culture and round spheroids in GrowDex,
while in monolayer culture the cells adhered tight to cell culture plastic and to each otherforming seamless layers of cells (Fig. 1A). In GrowDex cultures the formation of spheroids
occurred during the first day, resulting in symmetric, round and increasingly dense structures.
Cell viability increased modestly in both monolayer and GrowDex cultures, reflecting theslow growing nature of the cell line (Fig. 1B). This was also evident from imaging both thepHEMA and GrowDex cultures, where the cellular structures did not increase significantly insize across the 6 day study.

Figure 1. BT474 cells after 1, 3 and 6 din monolayer, suspension (pHEMA) andGrowDex cultures (A). In GrowDex cells
form multicellular tight spheroids (Lightmicroscope, 20x objective, scalebar
100 μm). BT474 viability in GrowDex
measured with CellTiterGlo shows 1.3fold increase from day 1 to day 6 (B).

COLO205 cells showed a typical phenotype of suspension cells failing to differentiate into
well-defined 3D structures (Fig. 2A). In the pHEMA coated wells the cells started to cluster
indicating cell-cell interactions at day 6. In GrowDex the cells formed small clusters, but notround well-defined spheroids like BT474. Cell viability assay indicated that in monolayer
culture there was a 3.4-fold increase from day 1 to day 6 (Fig. 2B). pHEMA culturedisplayed proliferation potential with a 6-fold increase from day 1 to day 6, in GrowDex theincrease was 2.3-fold. The slower proliferation rate in GrowDex may be related to the fiber
network affecting cell migration in the hydrogel.

Figure 2. COLO205 cells after 1,3 and 6 d in monolayer, suspension(pHEMA) and GrowDex cultures(A). In GrowDex the cells form small
multicellular structures (Light microscope,20x objective, scalebar 100 μm).
COLO205 viability quantified with
CellTiterGlo luminscence assay shows
2.3-fold increase from day 1 to day 6 inGrowDex (B).

CONCLUSIONSBoth cell lines BT474 and COLO205 were able to proliferate and form 3D multicellularstructures in GrowDex. BT474 is derived from solid breast ductal carcinoma tumor, while
COLO205 is derived from colorectal carcinoma ascites metastasis. BT474 showed theformation of round well-defined spheroids in GrowDex and loose cell clusters in suspension.
COLO205 expressed the typical phenotype of suspension cells without organized 3Dstructure. In GrowDex small multicellular structures were observed.
The results suggest that GrowDex can offer a suitable matrix for cancer cells derived bothfrom solid tumors and liquid biopsies. Nanofibrillar cellulose can be considered as a
biologically inert material, but the anchorage-dependency does not seem be a limiting factor
for 3D cell culture in GrowDex.

REFERENCES

1.
2.

3.

4.

Mori, S. et al. (2009). An Anchorage-Independent Cell Growth Signature Identifies
Tumors with Metastatic Potential. Oncogene, 28(31), 2796–2805.
Bhattacharya M. et al. (2012) Nanofibrillar cellulose hydrogel promotes threedimensional liver cell culture, Journal of Controlled Release 2012; 164:3, pp. 291–298
Lou Y.-R. et al. (2014) The use of Nanofibrillar cellulose hydrogel as a flexible threedimensional model to culture human pluripotent stem cells, Stem cell and development
2014; Vol. 23:4, pp. 380–392
Rinner B. et al. (2017) MUG-Mel2, a novel highly pigmented and well characterized
NRAS mutated human melanoma cell line. Scientific Reports 2017; 7, 2098

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