In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum

Abstract

Purpose: Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines.

Methods: The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS).

Results: Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1.

Conclusions: Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.

Fig. 1

The release of soluble mediators by human osteosarcoma cell lines cultured in serum-free and FCS-containing growth media. Seven different cell lines were examined: Saos-2, SK-ES-1, U2OS, Cal72, KHOS-321H, SJSA-1, and 143.98.2. The cells were cultured in the X-vivo 10 (X10), X-vivo 15 (X15), X-vivo 20 (X20), and Stem Span (SS) serum-free media (left part of each small figure), and in DMEM, F12 K medium, McCoy’s medium, MEM, RPMI 1640, and Stem Span medium supplemented with 10% FCS (right part). The levels were classified as undetectable and detectable levels corresponding to <50 pg/ml, 50–500 pg/ml, and >500 pg/ml (see upper right).

Fig. 2

PHA-stimulated T cell activation in the presence of osteosarcoma cells. Seven sarcoma cell lines (Saos-2, SK-ES-1, U2OS, Cal72, KHOS-321H, SJSA-1, and 143.98.2.) were cultured with the three different T cell clones CTC 4411 (▲), 4451 (●), and 4431 (■). Cultures were prepared in Stem Span medium with (+) or without (-) 10% heat-inactivated FCS, and the levels of IFNγ (right) and GM-CSF (left) were determined in the supernatants after 48 h of coculture in the presence of the T cell mitogen PHA. The results are presented as the incremental cytokine levels, i.e., the level in cultures containing PHA + CTC + sarcoma cells minus the summarised levels for corresponding control cultures containing CTC + PHA and CTC + sarcoma cells. Increased incremental levels are indicated by dark symbols, cultures without any increase are marked by open symbols. The clone 4434 was only cultured with Saos, SK-ES-1 and U2OS, and the figure therefore presents the results for 17 sarcoma-CTC combinations.