Penicillin g and streptomycin

Human bone marrow-derived mesenchymal stem cells (HBMSCs) were isolated according to the protocols described previously [37 (link)]. In brief, bone marrow aspirates were harvested from the greater trochanter of 14 femur fractures patients (8 men and 6 women, 25–48 years old) during surgery. Bone debris were removed by filtering and cells were then cultured in 75 cm² flasks at a density of 5.0 × 10⁵ cells/flask in the α-modification of minimum essential medium (α-MEM; Sigma-Aldrich, St Louis, MO, USA) containing 10 % fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1 % antibiotics (penicillin G and streptomycin, Gibco, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere containing 5 % CO₂. The medium was changed after 48 h to remove non-adherent cells and thereafter every 3 days. Cells were detached with 0.25 % trypsin–EDTA (Invitrogen, Rockford, IL, USA) and passaged at ~80 % confluence. Cells at the fifth passage were used for this study.
EaHy926, the human umbilical vein endothelial cell line, was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, St Louis, MO, USA) containing 10 % FBS and 1 % antibiotics at 37 °C in a humidified atmosphere containing 5 % CO₂. Cells were passaged at ~80 % confluence.

Human MSCs from six different donors were purchased from ALLCELLS and Lonza (Table 1). Because these cells were commercially available, no patient consent or approval from the FDA Research Involving Human Subject Committee was needed. Flow-cytometry analysis provided from each company revealed a marker profile of CD29+, CD44+, CD105+, CD166+, CD14-, CD34-, and CD45- for each donors’ MSCs. Cell viability was greater than 85% for all donors. MSCs were cultured in expansion medium containing αMEM (alpha minimum essential media) (Life Technologies, Grand Island, NY), 10% FBS (fetal bovine serum) (JM Bioscience, San Diego, CA), 1% L-glutamine (Life Technologies), and 1% penicillin G and streptomycin (Life Technologies) under a humidified atmosphere of 5% CO₂ at 37°C, according to Lo Surdo et al.[39 (link)]. After reaching 80% confluence, cells were detached from the flask by using 0.25% trypsin/1 mM EDTA solution (Life Technologies) and replated at a density of 60 cells/cm². The cells were expanded until the end of the seventh passage and snap frozen after passages 3, 5, and 7.

Passage 11 of Human gastric cancer cell line (AGS), Passage 15 of Human breast cancer cell line (MCF-7) and Passage 9 of murine B-16 melanoma cell line were obtained in 2014 as authenticated from national cell repository situated at National Centre for Cell Sciences (NCCS), Department of Biotechnology, Government of India, Pune, Maharashtra, India. The cells, initially seeded at the concentration of 10⁴cells/mm² were cultured at 37°C in humidified atmosphere of 5% CO₂–95% air. AGS cells were cultured in DMEM and F12K (Gibco, invitrogen, USA) whereas MCF-7 and B-16 melanoma were cultured in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum (FBS; Invitrogen.) and 1% penicillin G and streptomycin (Life Technologies, Rockville, MD, USA). Mycoplasma contamination detection was done using PlasmoTest (Invivogen. USA), all experiments were performed in Mycoplasma free cell lines.

Cell lines were obtained through ATCC and cultured with their recommending culturing protocol and media. Murine lymphoma EL4 (RRID: CVCL_0255) and human leukemia Jurkat T cells (RRID: CVCL_0367) were cultured at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 medium (Corning), respectively. Both cell culture media were supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM MEM nonessential amino acids, and 100 U/mL each of penicillin G and streptomycin (ThermoFisher).
Leukoreduction system white blood cells (LRS-WBC) were collected from the healthy donors from the San Diego Blood Bank (UCSD IRB Project #181004XX). cultured in RPMI-1640 media (Corning) supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, and 1 mM MEM nonessential amino acids (ThermoFisher).