4.2. Cell Lines and Culture Conditions (Standard Cell Culture Condition)

The cultivation of the DTC cell lines was essentially performed as described [32]. A detailed overview of the generation, authentication, and properties of the DTC cell lines BC-M1 (obtained from the bone marrow of a breast cancer patient), LC-M1 (obtained from the bone marrow of a lung cancer patient), and PC-E1 (obtained from the bone marrow of a prostate cancer patient) were reported before [20]. The DTC cell lines were cultured at 37 °C in a humidified environment with 5% of carbon dioxide and 10% of oxygen. The oxygen concentration was adjusted by N₂. The culture medium was RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mg/L insulin, 5.5 mg/L transferrin (all from Life Technologies, Darmstadt, Germany), 50 µg/L EGF (Miltenyi Biotec, Bergisch Gladbach, Germany) and 10 µg/L human basic fibroblast growth factor (b-FGF, Miltenyi Biotec). The bone metastatic sublines of MDA-MB-231, MDA-MB-231 SA [60], and MDA-MB-231 B02 [61] were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal calf serum (FCS) and 2 mM L-glutamine.

The cell lines by kind provision were: MDA-MB-231 B02 (Philippe Clézardin), MDA-MB-231 SA (Theresa A. Guise), Hs578t (Thomas Dittmar). MCF-7 (from ATCC, 2005; ATCC Cat# HTB-22), MDA-MB-231, MDA-MB-468 (DSMZ, Braunschweig, Germany, 09/2016; DSMZ Cat# ACC-738), BT20 (Cell Lines Service, Eppelheim, Germany, 2007; CLS Cat# 300130/p656_BT-20), BT549 (Cell Lines Service, 07/2008; CLS Cat# 300132/p770_BT-549), Hs578t, SCC25, Cal27, SKRBR3; they were cultivated in DMEM with 10% FCS and 2 mM L-glutamine (all from Life Technologies). The authenticated cell lines (last test) were MCF-7 (09/2017), MDA-MB-231 (02/2014), and Hs578t (09/2015). The authentication was performed by Multiplexion, Heidelberg, Germany by multiplex cell authentication (SNP-Profiling).

All of the cell lines were cultured at 37 °C in a humidified environment. The cell lines that were cultivated in RPMI were kept in the presence of 5% of CO₂, and the cell lines that were cultured in DMEM were kept in the presence of 10% CO₂. With the exception of the DTC cell lines, the remaining gas mixture was atmospheric air. These cell culture conditions are referred as to ‘standard cell culture conditions’ in this work. The cell lines were stored as cryo-cultures in liquid nitrogen, and the cells were temporarily resuscitated only for the experiments. After resuscitation, the cells were routinely tested for mycoplasmas using the VenorGeM Classic mycoplasma detection kit (Minerva Biolabs, Berlin, Germany, Cat. No. 11-1100). The mycoplasma test was performed after five passages (i.e., ±14 days), followed by the generation of fresh cyro-cultures if the cells were negative for mycoplasma. The mycoplasma-infected cells were expunged and the incubator was subjected to disinfestation. The experiments were performed from passage six to a maximum of 15 passages. The protein samples were generated within six months after the resuscitation of the cell lines.