Abstract
Purpose :
Three-dimensional (3D) in vitro systems allow for better physiological modeling than two-dimensional (2D) culture methods. The aim of this study is to evaluate the morphology, proliferation, and vascular endothelial growth factor (VEGF) production of uveal melanoma (UM) cell lines grown in different 3D-culture systems.
Methods :
Five UM cell lines were used: MEL270, OMM2.5, MEL285, 92.1, and MP41. Cells were grown using the traditional 2D system for adherent cells and six 3D-culture methods: anchorage-dependent (AD) techniques using cells admixed in media and methylcellulose (MC) as scaffold, on basement membrane extract (BME), and embedded in BME as a scaffold; and anchorage-free (AF) techniques using plates pre-coated with agarose (AG); simple hanging drop (HD); and modified hanging drop with MC (HDMC). Cultures were maintained for up to 96 hours and imaged with brightfield microscopy. CCK-8 cell counting was used to assess proliferation and ELISA to measure VEGF production. Image processing using ImageJ and statistical analysis using GraphPad Prism were performed.
Results :
Cells grown in 3D were observed to be pigmented when compared to 2D cultures. Tumor cells grown in anchorage-dependent (AD) conditions formed heterogeneous aggregates that were morphologically distinct across cell lines. Proliferation rates were lower when cells were cultured on BME versus 2D and decreased after 96 hours. Spheroids were formed with the AF techniques; HDMC provided consistent compaction after 48 hours. Based on area and circularity, the optimal seeding density was at 10,000 cells/20 μL per drop, although spheroids formed with as low as 500 cells/20 μL drop. The average area of spheroids after 48 hours ranged from 0.17±0.01 mm2 to 1.87±0.09 mm2. Circularity but not roundness significantly differed across cell lines. VEGF production differed among cell lines and the use of AG plates induced low VEGF levels even under hypoxia.
Conclusions :
This is the first study to characterize multiple UM cell lines grown in various 3D-culture systems. A BME matrix may be used to form heterogeneous aggregates. To form UM spheroids, the modified hanging drop with MC was most reliable. These techniques offer better alternatives when studying UM pathophysiology and testing potential therapeutic agents. Additional cellular parameters should be characterized for these 3D-culture systems.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.