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. 2016 Jun;45(3):219-30.
doi: 10.1111/ahe.12190. Epub 2015 Aug 19.

Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures

C Gebhard  1 , C Gabriel  1 , I Walter  1   2
Affiliations

Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures

C Gebhard et al. Anat Histol Embryol. 2016 Jun.

Abstract

Spheroid 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.

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Figures

Figure 1
Figure 1
Examples of cell culture phase contrast light microscopy images of D17 canine osteosarcoma cells cultivated as 2D monolayers (a, b) or three‐dimensional (3D) spheroids (c, d). Monolayer cultivated to near confluence after 2 days. Within the monolayer lawn, epithelial‐like cells were mainly observed; occasional spindle‐shaped cells were located within less confluent areas (a). 2D monolayer after 7 days exhibited an extremely dense cell lawn (b). Spheroids developed the characteristic 3D architecture as round‐shaped cell conglomerates within the first 7 days of cultivation (c). After cultivation for 14 days, spheroids exhibited increased diameters with tightly arranged cells (d). Scale bars = 200 μm.
Figure 2
Figure 2
Histochemical and immunohistochemical staining on 2D monolayer (a–h) and three‐dimensional spheroid (e–t) cell culture. The illustrated samples were stained for H&E (a, e, j, m, q) as well as immunostained for Ki‐67 proliferation marker (b, f, j, n, r), osteocalcin (c, g, k, o, s) as an indicator for calcification and collagen I (d, h, l, p, t) for fibre demonstration. Scale bars = 100 μm.
Figure 3
Figure 3
Histochemical analysis of sections of two‐dimensional (2D) monolayer cell cultures (a, c, e, g) and three‐dimensional (3D) spheroids (b, d, f, h). Samples were stained for aniline blue (a, b) and periodic acid–Schiff (PAS) (c, d). Note the delicate network of extracellular matrix positive for PAS and aniline blue, particularly in 3D spheroids. Immunohistochemical detection of S100A4 (e, f) and ezrin (g, h) in 2D and 3D cultures. Scale bars = 100 μm.
Figure 4
Figure 4
Transmission electron microscopy images of two‐dimensional (2D) monolayers and three‐dimensional (3D) spheroids generated from canine osteosarcoma cells (D17). Characteristic 2D monolayer cells cultivated for 2 days show tight cell contacts lacking signs of extracellular matrix structures between cells (a, b). Scale bars = 5 μm. Inter‐cellular space marked by arrowhead (b). Note viable tumour cells with characteristic nuclei with the prominent nucleoli in spheroids cultivated for 7 day (c). Inter‐cellular space provided with collagen; arrowheads (c, d). Scale bar = 2.5 μm. Undefined electron‐dense agglomerates at the cell membranes with tight cell–cell contacts within the spheroids (black arrow) in spheroids after 14 day in culture (e). Collagen I fibres were clearly identified in inter‐cellular space of spheroids cultivated for 14 day (f). Scale bar = 0.5 μm. After 19 days of culture, the amount of lipid droplets as well the amount of necrotic cells (black arrowhead) was increased (g). Scale bar = 2.5 μm. Densely arranged collagen I fibres predominated the extracellular space in 19‐day spheroids (h). Scale bar = 0.5 μm.
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References

    1. Akeda, K. , Nishimura A., Satonaka H., Shintani K., Kusuzaki K., Matsumine A., Kasai Y., Masuda K., and Uchida A., 2009: Three‐dimensional alginate spheroid culture system of murine osteosarcoma. Oncol. Rep. 22, 997–1003. - PubMed
    1. Arai, K. , Sakamoto R., Kubota D., and Kondo T., 2013: Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system. Proteomics 13, 2351–2360. - PubMed
    1. Bae, H. , Chu H., Edalat F., Cha J. M., Sant S., Kashyap A., Ahari A. F., Kwon C. H., Nichol J. W., and Manoucheri S., 2014: Development of functional biomaterials with micro‐and nanoscale technologies for tissue engineering and drug delivery applications. J. Tissue Eng. Regen. Med. 8, 1–14. - PMC - PubMed
    1. Bartel, C. , Tichy A., Schoenkypl S., Aurich C., and Walter I., 2013: Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium. BMC Vet. Res. 9, 86. - PMC - PubMed
    1. Bissell, M. J. , 1981: The differentiated state of normal and malignant cells or how to define a “normal” cell in culture. Int. Rev. Cytol. 70, 27–100. - PubMed

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