Trypsin

Mononuclear cells were isolated from BM aspirates by density gradient centrifugation using Lymphoprep technology (STEMCELL), and after CD34⁺ purification, CD34⁻ cells were plated in noncoated 75–150 cm² tissue culture flasks (BD Falcon) at a density of 1 × 10⁵/cm² in complete culture medium: DMEM low glucose (Gibco, Life Technologies) supplemented with 5% platelet lysate (PL), 1% penicillin/streptomycin (Pen/Strep, EuroClone), and 2 mM l‐glutamine (l‐Glu, EuroClone). After 48 hr in culture, nonadherent cells were removed and culture medium was replaced twice a week. MSC were harvested, after reaching ≥80% confluence, using Trypsin (EuroClone), and were propagated at a density of 4x10³ cells/cm². MSC at passage 2–4 (P2–P4) were considered “early passages” MSC, while MSC at passage 10 (P10) were considered “late passages” MSC.

The in vitro cytotoxicity effects of the MSN particles in Caco-2 cultures was assessed by counting the cell number using the improved Neubauer hemocytometer chamber and an optical microscope [34 (link),46 (link),47 (link),48 (link)]. Prior to each test, the cells were harvested using trypsin (Euroclone)–ethylenediamine tetraacetic acid (EDTA, Sigma–Aldrich)–phosphate-buffered saline (PBS) solution (0.25% trypsin–0.05 mM EDTA) and diluted at a density of 10⁵ cells/mL. The cell suspension was seeded into 96-well plates (Corning Inc., Corning, NY, USA) at 100 μL/well. After cell attachment (3–4 h), the material suspensions of MCM-41, aprepitant-loaded MCM-41, MCM-48, and aprepitant-loaded MCM-48, with increasing concentration (100, 500, and 2000 μg/mL), were added to the cultures and cells were incubated for 48 h. Subsequently, cells were detached by trypsinization and the number of cells in culture was measured using the Neubauer chamber under the microscope in an attempt to evaluate the effect of the MSN particles on cell proliferation. Simultaneously, the cellular death in Caco-2 cultures was assessed using the trypan-blue dye exclusion method [34 (link),48 (link)]. Then cell growth capacity in treated cultures was expressed as a percentage (%) of that observed in the untreated control.

Vero cells (African green monkey kidney cell line, ATCC), 3T3 BALB/c cells, a fibroblast cell line derived from BALB/c mice, and human HeLa3T1 cells [50] (link), containing an integrated copy of plasmid HIV-LTR-CAT in which expression of the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the HIV-1 LTR promoter, were grown in DMEM (Euroclone, Grand Island, NY). The mouse dendritic cell line CB1 [51] (link) was grown in ISCOV (Euroclone). Media were supplemented with 10% FBS (Euroclone), 1% L-glutamine (100X solution, 0.2 M, BioWhittaker, Walkersville, MD), 1% penicillin/streptomycin (100X solution, Euroclone). The cells were detached with trypsin solution containing 0.25% trypsin and 0.02% EDTA (Euroclone). Splenocytes from immunized and control mice were cultured in RPMI 1640 (Euroclone) supplemented with 10% Hyclone (Euroclone), 50 µM β-mercaptoethanol (Gibco, Grand Island, NY), 1% L-glutamine, 1% penicillin/streptomycin, 1% non-essential aminoacids (Sigma-Aldrich, St. Louis, MO) and 1 mM sodium pyruvate (Sigma-Aldrich) (referred to as RPMI1640 complete medium). All cells were grown at 37°C in 5% CO₂.

Primary skin fibroblast cell lines were isolated from skin biopsies as follows. Skin specimens were washed with 70% ethanol and physiological solution and incubated with 2 mg/mL Dispase II (Merck KGaA, Darmstadt, Germany) overnight at 4 °C. Then, the dermis was separated using sterile tweezers, cut into 2–4 mm pieces and plated on a 6-well microplate. A squared sterile glass was put above and 1 mL of high glucose Dulbecco’s modified Eagle’s medium, DMEM (Euroclone, Milan, Italy) supplemented with 20% fetal bovine serum, FBS (Euroclone, Milan, Italy) was added to each well. After 3 weeks, dermis and glasses were removed and fibroblasts were detached with 0.25% Trypsin and 0.02% EDTA (Euroclone, Milan, Italy) and plated in 25 cm² flasks. Cells were then cultured in DMEM supplemented with 15% (v/v) FBS, 100 U/mL penicillin, 100 mg/mL streptomycin (Euroclone, Milan, Italy) and 2 mM L-glutamine (Euroclone, Milan, Italy) and maintained at 37 °C under humidified conditions and 5% CO₂. Cells were sub-cultured twice weekly, detached with Accutase (Euroclone, Milan, Italy) and centrifuged at 500× g for 10 min at room temperature. Cells were used for proteomics analysis at passage number lower than 10. Cell pellets were collected, washed in PBS, frozen in liquid nitrogen and stored at −80 °C.

We used SKBR3 cells (ICLC, Genova, Italy), that are the reference HER2+, human, BC epithelial cell line 28 (link) and MCF10A cells, as human, normal-like epithelial breast cells 29 (link). We maintained the cell line within a humidified atmosphere containing 5% CO₂ at 37 °C in DMEM high glucose cell culture medium (Gibco, Life Technologies), with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2mM L-Glutamine (Euroclone) following the manufacturer's recommendation. Dulbecco Phosphate-Buffered Saline (D-PBS), and trypsin were obtained by Lonza (Euroclone).

The human embryonic kidney cell line 293T and the human Ewing sarcoma cell line A673, both from our laboratory, were maintained in culture at 37 °C in Dulbecco’s Modified Eagle’s Medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories Inc., Etobicoke, Canada), 1% Penicillin/Streptomycin (10,000 U/mL Penicillin, 10 mg/mL Streptomycin in 0.9% NaCl solution, PAA Laboratories Inc.). Adipose-derived MSC (AD-MSC) were obtained as previously described from lipoaspirate specimens of individuals undergoing liposuction for esthetic purposes after approval by local Ethical Committee [7 (link)]. Cells from two different donors were used as biological replicates. After isolation, cells were grown in α-MEM (Gibco) containing 2.5% human platelet lysate (Modena Policlinic Blood Bank, Modena, Italy), 1% L-Glutamine (200 mM in 0.85% NaCl solution, BioWhittaker, Lonza, Verviers, Belgium), 0.5% Ciprofloxacin (Fresenius Kabi Italia S.r.l., Verona, Italy), 1 IU/ml Heparin (Sigma-Aldrich, St. Louis, MO, USA). When confluent, the adherent AD-MSC cells were detached with Trypsin/EDTA (Trypsin 0.05% EDTA 0.02% in PBS, EuroClone, Milan, Italy), counted and seeded at 6000 cells/cm². Cells were incubated and maintained within a controlled atmosphere (5% CO₂ and temperature of 37 °C).

Murine macrophages (J774 A.1 cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Euroclone) supplemented with 10% inactivated fetal bovine serum (FBS) (Euroclone, Italy), 1% L-glutamine (Euroclone) and 1% streptomycin–penicillin (Euroclone) and were incubated at 37 °C and 5% CO₂. Adherent cells were washed with sterile warm phosphate buffered saline (PBS) (Euroclone) and removed for experiments by using 1× trypsin in PBS (Euroclone). Cells were counted and re-suspended in DMEM supplemented with 2% FCS and 1% L-glutamine. Finally, cells were seeded in sterile 48 well plates (Euroclone) at a concentration of 1.2 × 10⁶ cells/well and incubated overnight until infection or treatment [10 (link),23 (link)].