Abstract
SummarySpheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold‐free three‐dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19‐day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14‐ and 19‐day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki‐67 immunoreactivity showed an even distribution in two‐dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis‐associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold‐free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell–cell and cell–matrix interactions.
Highlights
Osteosarcoma (OS) is the most common primary bone tumour in adolescents and young adults (He et al, 2014; Xing et al, 2014)
OS frequently arises in the skeletal region at the medullary side of long bones as well as in lower numbers at the extraskeletal region (Klein and Siegal, 2006; Broadhead et al, 2011; Gill et al, 2013; Luetke et al, 2014). This aggressive bone tumour is combined with severe clinical symptoms such as joint pain, heavily decreased mobility, fracture and metastatic spread (Picci, 2007; Clark et al, 2008)
In the first 2 days, D17 cells displayed partly epithelial-like shaped cells and partly spindle-shaped morphology which developed until day 7 into a very dense epithelial-like cell lawn with areas of overgrowing cells (Fig. 1b)
Summary
Osteosarcoma (OS) is the most common primary bone tumour in adolescents and young adults (He et al, 2014; Xing et al, 2014). OS frequently arises in the skeletal region at the medullary side of long bones as well as in lower numbers at the extraskeletal region (Klein and Siegal, 2006; Broadhead et al, 2011; Gill et al, 2013; Luetke et al, 2014) This aggressive bone tumour is combined with severe clinical symptoms such as joint pain, heavily decreased mobility, fracture and metastatic spread (Picci, 2007; Clark et al, 2008). Monolayer cultures are maintained as flattened cells adhered on plastic surfaces These two-dimensional (2D) models mirror an unnatural morphology and are likely to have considerable influence on drug response. There is an increasing awareness of these drawbacks of 2D cell cultures (Hyman and Simons, 2011; Tan et al, 2011; Prideaux et al, 2014)
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