Fetal bovine serum (fbs)

Mouse cancer cell lines were maintained in Dulbecco’s Modified Eagle Medium (Sigma, United States) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Germany), 1 mM sodium pyruvate (Gibco, United States), 1× MEM non-essential amino acid solution (Sigma, United States), and 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, United States).
CHO-Flt3L cells were maintained in Iscove’s Modified Dulbecco’s Medium (Sigma, United States) supplemented with 5% heat-inactivated fetal bovine serum (PAA Laboratories, Germany).
Sp37A3 mouse dendritic cell line and relative genetically modified lines were maintained in Iscove’s Modified Dulbecco’s Medium (Sigma, United States) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Germany), 1 mM sodium pyruvate (Gibco, United States), 1× MEM non-essential amino acid solution (Sigma, United States), 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, United States), 0.05 mM 2-mercaptoethanol (Gibco, United States), 20 ng/ml recombinant mouse GM-CSF (Peprotech, United Kingdom), and 20 ng/ml recombinant mouse M-CSF (Peprotech, United Kingdom).

Mouse brain microglial cells (BV-2) were cultured with RPMI 1640 (Hyclone™, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (GE Healthcare Life Sciences) and 1% penicillin-streptomycin solution (Thermo Fisher Scientific). A mouse macrophage cell line (RAW 264.7) was purchased from the Korean Cell Line Bank (KCIB, Seoul, Korea) and cultured with Dulbecco’s Modified Eagle Medium high glucose cultured media (Hyclone™, GE Healthcare Life Sciences), 10% fetal bovine serum (GE Healthcare Life Sciences), and 1% penicillin-streptomycin solution (Thermo Fisher Scientific). The cultured cells were incubated in a humid atmosphere under 5% CO₂ at 37 °C.

3D co-cultures of PBMC with MSC (n = 6) were performed in 12-well plates (Corning). Cells were embedded in fibrin matrices in a ratio 1:100 (MSC:PBMC) using 2.5 × 10⁶ PBMC/cm² and cultured for up to 2 weeks in complete αMEM medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences). Fibrin matrices were prepared as described above, but without addition of aprotinin. Control experiments were performed culturing PBMC separated from MSC by a 0.4 μm transwell insert (Corning), PBMC without support of stromal cells and MSC alone. To investigate the effect of paracrine inflammatory signals on stromal cells in the absence of immune cells, MSC (n = 4) were embedded in fibrin matrices in 24-well plates (Corning) at 2.5 × 10⁴ cells/cm² and cultured for 6 days in complete αMEM medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences) supplemented with 5 ng/ml tumor necrosis factor (TNF)α (PeproTech, Rocky Hill, NJ) and 10 ng/ml interferon (IFN)γ (PeproTech). Control experiments were performed in complete αMEM medium without cytokine supplementation. Cultures were maintained at 37°C (20% O₂ and 5% CO₂ humidified atmosphere), and medium was changed every 3 days. Cellular re-arrangement was monitored using a phase contrast microscope (Olympus) and documented using a digital camera (Olympus).

Cells were maintained at 37 °C in a 5% CO₂ atmosphere. hTERT RPE-1 cells (ATCC cat. no. CRL-4000, RRID:CVCL 4388) and HEK 293 cells (ATCC cat. no. CRL-1573, RRID:CVCL 0045) were grown in Dulbecco’s modified medium (DMEM) F12 (11320-033 from Gibco) containing 10% fetal bovine serum (GE Healthcare), 100 U ml⁻¹ penicillin, 100 U ml⁻¹ streptomycin (15140-122 from Gibco). BJ cells (ATCC cat. no. CRL-4001, RRID:CVCL 6573) and HCT116 cells (ATCC cat. no. CCL-247, RRID:CVCL 0291) were grown in Dulbecco’s modified medium + GlutaMAX (61965-026 from Gibco) containing 10% fetal bovine serum (GE Healthcare), 100 U ml⁻¹ penicillin, 100 U ml⁻¹ streptomycin (15140-122 from Gibco).
All cells were routinely checked for mycoplasma infection and are negative for mycoplasma infection. Identity and purity of the human cell lines used in this study were tested and confirmed using STR authentication.