Ccl 75tm

WI-38 human fetal lung fibroblasts (ATCC, CCL-75TM, LOT 58110309) were maintained in HyClone minimum essential medium (MEM) Alpha Modification (ThermoScientific) containing 10% (v/v) fetal bovine serum (FBS; Gibco) in a humidified Forma Steri-Cycle CO₂ Incubator (ThermoScientific) containing 6% CO₂ at 37°C. WI-38 cells have a replicative limit of 50 ± 10 population doublings (PDs). In this study, cultures of three different PDs were used—38 (young), 47 (intermediate), or 54 (old), as previously described (Sidler et al., 2014 (link)). The PD number of a culture is the sum of all ΔPD = log₂(n_f/n_i) for each passage, where n_f is the final number of cells in a passage and n_i is the initial number of cells inoculated.

Human epithelial breast cancer cell line MCF7 (ATCC^® HTB-22^TM, ATCC, Manassas, United States) and human mesenchymal fibroblast cell line Wi-38 (ATCC^® CCL75^TM, ATCC) were purchased directly from ATCC. The cells were propagated in culture using the AMEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, United States) with 5% bovine serum, glutamine (10 Mm, Gibco), crystacillin (100 U/ml; Pliva d.d., Zagreb, Croatia) and gentamicin (50 μg/ml; Krka d.d., Novo mesto, Slovenia). The cells were cultured in a 5% CO₂ humidified incubator at 37°C. The cells were grown as a monolayer until they reached at least 80% confluence. Afterward, the medium was removed, the cells were first washed with PBS and afterward detached from the surface with 0.25% trypsin/EDTA in Hank’s buffer (Gibco). After detachment, the cells were collected, counted, and a cell suspension with appropriate cell density was prepared. The cultured cells (100 or 1000 cells) were spiked into 10 ml of diluted BC and processed within 1 h identically to the patients’ whole blood specimens. Altogether, 14 BC samples were spiked with MCF7 cells and eight samples were spiked with Wi-38 cells.