Application Note 18

Novel NRAS mutated humanmelanoma cell line MUG-Mel2
3D culture in GrowDex®
Beate Rinner1, Johanna Niklander2, Raili Koivuniemi2, Marjo Yliperttula2
1
Medical University of Graz, Austria
2
University of Helsinki, Finland

INTRODUCTION

NRAS mutation in melanoma is associated with aggressive tumor biology and poor
prognosis. Therapy response rates for NRAS mutated melanoma are significantly weaker
than for BRAF mutated melanoma. Well-characterized NRAS mutated cell lines with correct
phenotype are needed for research and drug development. While plenty of cell lines exist,
only few melanogenic cell lines retain their in vivo characteristics.
In this application note, 3D culture of novel NRAS mutated human melanoma cell line
MUG-Mel2 in GrowDex® is presented. MUG-Mel2 was derived from a patient withaggressive cutaneous ulcerated melanoma. The 3D culture matrix was required to be tuned
for MUG-Mel2 due its novel status, and the scaffold needed also to be removable for further
downstream analysis. In previous studies GrowDex has been shown to be suitable 3D culture
matrix for various cell types, supporting also organotypic growth, due to the tunability of themechanical properties of the hydrogel. GrowDex can be also removed by simple cellulase
enzyme treatment.

MATERIALS• MUG-Mel2 cell line (Medical University of Graz)• RPMI cell culture medium with 10% FBS, 2mM L-glutamine, 1% penicillin/ streptomycin(Cat No. 11875-093, Life Technologies)• GrowDex 1.5% (Cat No. 100 300 105, UPM)
• GrowDase 10mg/ml (Cat No. 900 100 102, UPM)
• Cellstain double staining kit (Cat No. 04511, Sigma)• DAPI (Cat No. D1306, Invitrogen)• Prolong Diamond Antifade Mountant (Cat No. P36965, Life Technologies.

METHODSRESULTS1.

To determine the optimal conditions for 3D cell culturing, three concentrations (0.4%;0.7% and 1% wt/v) of GrowDex, as well as two cell seeding densities (50,000 and100,000 cells per 100 µl of GrowDex/96-well) were tested.
2. MUG-Mel2 spheroids grown in GrowDex were collected at day three for live calcein
staining. Prior to collection, GrowDex was degraded from the cell culture by GrowDase(UPM) cellulase enzyme treatment for 24 h at 37°C using 300 µg GrowDase per 1 mgof cellulose in GrowDex.
3. Confocal imaging:a. Spheroids were washed three times with 1xDPBS, suspended in staining solution
containing Calcein-AM 1:500 (Cellstain double staining kit, Sigma) and 50 µg/ml DAPI (Invitrogen) for 15 min at 37°C, and subsequently mounted using Prolong
Diamond Antifade Mountant (Life Technologies).
b. Fluorescent images were taken with the Leica TCS SP5 microscope using HCX PL
APO 20x/0.7 objective.

MUG-Mel2 cells were able to form multicellular spheroid structures after one day culturein GrowDex. The optimal condition for the spheroid formation was found to be between
0.4% and 0.7% GrowDex concentration with cell seeding density of 50,000 cells/ml. In
1% GrowDex MUG-Mel2 cells did not form spheroids. After three days in culture, MUGMel2 formed spheroids with diameter around 200 µm, showed good viability and correct
morphology (Fig. 1 A-C). High pigmentation typical for melanoma cells was clear after fivedays culture in GrowDex (Fig. 1D).

Figure 1. Morphology of MUG-Mel2 cell spheroids in GrowDex after three days culture
in a concentration of 0.5% (A,B). Calcein staining (green) revealed the viability of thespheroids, nuclei were counterstained with DAPI (blue) (C). After five days in GrowDex,
MUG-Mel2 showed intensive brown staining of the cell cytoplasm typical for melanoma
cells (D).

CONCLUSIONSREFERENCES

The newly established cell line MUG-Mel2 cultured in GrowDex offers a promising model
for NRAS mutated melanoma research and therapy development. GrowDex presents anoptimal matrix for the cells to maintain their natural behavior and the unique phenotype ofthe melanoma - the stable brown coloring. Cell culture models that maintain characteristicsof the in vivo state are important research models to explore new and relevant targets formelanoma treatment.

1.

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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