Chemically defined formation of spheroids loaded with superparamagnetic iron oxide nanoparticles

J. Wiest1*, E. Stetter2, M. Koch3

11th World Congress on Alternatives and Animal Use in the Life Science, Abstract overview, 2020, 60

1 cellasys GmbH, Kronburg / Germany, 2 Stetter Elektronik, Seeheim-Jugenheim / Germany, 3 Feldkraft Ltd., Copenhagen / Denmark

Corresponding author: J. Wiest, wiest@cellasys.com

To increase physiological relevance and to develop new therapies 3D spheroids are of increasing interest. The addition of superparamagnetic iron oxide nanoparticles (SPIONs) into the spheroid allows for investigation of new therapeutic strategies (Zhang et al., 2014) without the use of animals (Lei and Schaffer, 2013; Whatley et al., 2014; Marx et al., 2016). L929 fibroblasts were maintained in 25 mL Greiner Bio-One AdvancedTC© cell culture flasks in chemically defined DME/F12+ITS cell culture medium. Cells received fresh medium twice a week and were passaged at about 80% confluency once a week. Spheroids were created similar to a previously developed method (Alexander, Eggert and Wiest, 2018). The 20 nm SPIONs beads were obtained from micromod Partikeltechnologie GmbH (Rostock / Germany) and a stock solution with 100 µg/mL was prepared. Spheroids were prepared in a 96 well-plate with cell-repellent surface (Greiner Bio-One GmbH, #650970). Each well was filled with 190 µL DME/F12+ITS containing 10.000 L929. For the SPION loaded spheroids (SPION-LS), 10 µL of magnetic beads stock solution was added, whereas for the non-magnetic control spheroids (NM-CS) 10 µL of cell culture medium was added. Then the 96well plate was centrifuged at 1000 g for 5 min and finally incubated at 37°C with 5% CO2. 100 µL of medium in each well was replace by fresh DME/F12+ITS (preheated to 37°C) daily. Spheroids were used for the experiments on day 5. For manipulation of the spheroids a neodym magnet (length 25 mm, diameter 25 mm) was used in a distance of approximately 30mm. The calculated field force acting on the spheroids was approximately 30mT. To investigate if the SPIONs are incorporated into the spheroids one SPION-LC was transferred to a well with one NM-CS. The movement of the SPION-LC due to the applied magnetic field can be seen at https://youtu.be/ev9bZnKyvGA .

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Lei, Y. and Schaffer, D. V. (2013) ‘A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation’, Proceedings of the National Academy of Sciences of the United States of America, 110(52). doi: 10.1073/pnas.1309408110.

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Whatley, B. R. et al. (2014) ‘Magnetic-directed patterning of cell spheroids’, Journal of Biomedical Materials Research – Part A, 102(5), pp. 1537–1547. doi: 10.1002/jbm.a.34797.

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