Application Note 30

Development of 3D osteosarcoma/
fibroblast co-culture model using
nanofibrillar cellulose
Markus Rimann1, Adem Regez1, Lauri Paasonen2
1
Zurich University of Applied Sciences ZHAW, Wädenswil, Switzerland
2
UPM-Kymmene, Finland

INTRODUCTIONIn drug research and development traditional two-dimensional (2D) in vitro cell models
are commonly used to determine e.g. the drug efficacy. Unfortunately 2D models, andalso in vivo experiments, are often unreliable and show different results than in humans.
It is important to have representative in vitro models available already in the preclinicalphase. Three-dimensional (3D) cell culture enables more functional cell models compared
to traditional 2D models (1,2). Co-culturing of more than one cell type in 3D environment
presents more physiologically relevant conditions due to interactions between different celltypes of the tissue.
In this study we present a 3D co-culture model of osteosarcoma cells (SaOS-2) with humandermal fibroblasts (HDF) in nanofibrillar cellulose hydrogel, GrowDex®.

MATERIALS• GrowDex 1.5% (Cat No. 100 100 005, UPM)
• DMEM/F12 supplemented with 1% penicillin/streptomycin, 10% fetal bovine serumand 200 mM L-glutamine (Sigma-Aldrich)• SaOS-2 cells (Acc-243, DSMZ)• Human dermal fibroblast cells (CellnTEC)• 96-well Ultra Low Attachment (ULA) plates (Cat No. 3474, Corning)• CellTiter-GLO Luminescent Cell Viability Assay (Cat No. G7572, Promega)

METHODS• GrowDex concentration 0.5% was used for the cultures• Cell seeding density was 625,000 cells/ml• Both cell types were cultured separately, followed by the co-culturing• Tested co-culture ratios were 1:1, 1:2, 1:4, 1:6 and 1:8 (SaOS-2:HDF)• Total number of cells remained constant, only ratio between the cell types waschanged
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RESULTS AND DISCUSSION

Figure 1. Comparison of themonocultures after 14 days of cultivation
in 0.5% GrowDex. A) SaOS-2 cultureand B) HDF culture. The HDFs form small,compact and spherical microtissues,
while the SaOS-2 form loose andgrape-shaped microtissues. Scale bar is
100 μm.

Figure 2. SaOS-2:HDF co-culture
morphologies at a ratio of 1:4 (SaOS2:HDF) after 14 days of culture in 0.5%
GrowDex. The microtissues form different
morphologies: SaOS-2 -like morphology(A), typical HDF -like morphology(B), and HDF -like morphology withadditional cell assemblies around thecore (C). Scale bar is 100 μm.

GrowDex was diluted to 0.5% with medium by mixing with a low retention pipette
Cell suspension was added and mixed by pipetting
50 μl GrowDex/cell mix was transferred into 96-wellplate and 100 μl medium wasadded on top of the hydrogel
Cultures were incubated in 5% CO2 at 37°C
• Half of the medium was replaced every two to three days
Cell morphology and size distribution were analysed by light microscopy with Axiovert
25 microscope and AxioVision Rel. 4.8 software
Cell viability was quantified by CellTiter-GLO luminescent assay• 70 μl medium was removed. GrowDex with the medium was transferred in 		Eppendorf tube by pipetting• 30 μl CellTiter-GLO reagent was added to the samples and shaken 20 min with300 rpm at room temperature• Samples were centrifuged at max. speed for 30 sec• 40 μl of the supernatant was transferred in the 96-well half-area luminescenceplate and measured with Optima FLUOStar plate reader

Both cell types form 3D structures in GrowDex when grown in monoculture. SaOS-2 cells are
more likely to form loose cell aggregates (Fig. 1A) and the HDF cells form compact spheroids(Fig. 1B). HDF spheroids reached maximum size after 7 days culture, and subsequently thesize decreased. The SaOS-2 did not show size decrease over time. Co-cultured microtissues
showed three different morphologies: a similar compact structure to HDF (Fig. 2A), loose
microtissues similar to the SaOS-2 (Fig. 2B), and a compact core with loose cells aroundthe core (Fig. 2C). The size distributions of co-cultured microtissues with different cell ratios
at culture days 4, 7, and 14 are presented in Fig. 3. The microtissues showed smaller sizewith higher HDF cell numbers, which could be due to the inhibitory effect of HDF cells on thetumor cells (SaOS-2) (3).

Figure 3. Size distribution of SaOS2:HDF co-culture microtissues at ratios1:1, 1:4, and 1:8 over a cultivation
period of 14 d in 0.5% GrowDex.

RESULTS AND DISCUSSION

SaOS-2 and HDF were successfully co-cultured in GrowDex with different cell ratios.
Microtissues showed different morphologies. Smaller microtissues were formed with higher
HDF cell numbers, likely due to the inhibitory effect of HDF cells on the tumor cells. Further
histological studies of the microtissues is required to analyze the cell distribution of SaOS-2and HDF in the co-cultures. Co-culturing of SaOS-2 tumor cells with stroma cells (HDF) in 3Dhydrogel results in physiologically more relevant microtissues that could be used as an in vitro
model e.g. in osteosarcoma drug development.

REFERENCES

M. Rimann and U. Graf-Hausner, “Synthetic 3D multicellular systems for drug development,”
Curr. Opin. Biotechnol., vol. 23, no. 5, pp. 803–809, 2012
M. Rimann et al., “An in vitro osteosarcoma 3D microtissue model for drug development,” J.
Biotechnol., vol. 189, pp. 129–135, 2014
A. Sadlonova et al., “Breast fibroblasts modulate epithelial cell proliferation in threedimensional in vitro coculture,”Breast Cancer Res., vol. 7, no. 7, p. R46, 2005

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