2.10. Cell Culture

Murine cancer associated fibroblasts (CAFs) were isolated from 4T1 breast tumors generated as described below in Section 2.16. After 21 days of tumor growth, tumors were excised and dissociated using a tumor dissociation kit (Miltenyi Biotec S.r.l., Bologna, Italy; catalogue number: 130-096-730). From the single cell suspension, CAFs were isolated using the tumor-associated fibroblast isolation kit (Miltenyi Biotec S.r.l., Bologna, Italy; catalogue number: 130-116-474) according to the manufacturer protocol. Briefly, this includes a first incubation of the dissociated tumor with magnetic beads for non-tumor associated fibroblasts depletion followed by the positive selection of CD90.2-positive tumor associated fibroblasts. To check the isolation yield, the cells were fluorescently stained with CD45-FITC and CD90.2-PE antibodies and analyzed by CytoFLEX flow cytometer (Beckman Coulter, Cassina De’ Pecchi, Italy). Isolated CAFs were cultured in DMEM/Ham’s F-12 medium supplemented with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1% non-essential amino acids.

Primary human activated myofibroblasts (HMfs) were isolated from patients with Crohn’s disease undergoing ileal surgical resection, as previously described [40]. Briefly, a biopsy of strictured intestinal mucosa was digested by incubation with 1 mg/mL collagenase A (Sigma-Aldrich S.r.l., Milan, Italy), 50 ng/mL DNase I (Sigma-Aldrich S.r.l., Milan, Italy) in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics for 2 h at RT. After filtration through Cell Strainer 100 μm (BD Biosciences, San Jose, CA, USA), isolated myofibroblasts were seeded in tissue culture flasks and maintained in DMEM supplemented with 20% FBS, 2 mM L-glutamine, 200 U/mL penicillin, 0.2 mg/mL streptomycin, 1% non-essential amino acids, 0.25% gentamicin, and 0.4% amphotericin B. Collection of human samples was authorized by the Ethical Committee of ASST Fatebenefratelli Sacco university hospital (protocol number 0002846). All the patients signed a written informed consent prior to their inclusion in the study.

Immortalized breast cancer murine 4T1-Luc2 (Bioware Ultra, Perkin Elmer, Milan, Italy) and human MDA-MB-231 (HTB-26, ATCC-LGC Standards, Sesto San Giovanni, Italy) were cultured in RPMI 1640 and in high glucose Dulbecco’s Modified Eagle Medium (DMEM) respectively. Both media were supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.

All cells were grown at 37 °C in a humidified atmosphere containing 5% CO₂ and were subcultured prior to confluence using trypsin/EDTA. Cell culture media and chemicals were purchased from Euroclone.