Penicillin streptomycin antibiotic

Human ampullary adenocarcinoma cell lines including SNU-869 and SNU-478 were obtained for Chinese Academy of Sciences (Shanghai, China). All cell lines were routinely tested for mycoplasma contamination and authenticated by Short Tandem repeat (STR) profiling. Cells were maintained in recommended medium, Roswell Park Memorial Institute-1640 (RPMI-1640, Corning) or DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS, Sigma‐Aldrich) and 1% penicillin–streptomycin antibiotic (Sigma‐Aldrich) and incubated at 37 °C and 5% CO₂ in a humidified atmosphere in an incubator.

Dimethyl sulfoxide (DMSO), Dulbecco’s
Modified Eagle Medium (DMEM), Dulbecco’s phosphate buffer saline
(PBS), Fetal bovine serum (FBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), l-glutamine, penicillin/streptomycin antibiotics,
sodium pyruvate, trypan blue and trypsin-EDTA solutions were all obtained
from Sigma-Aldrich (St. Louis, MO). LysoTracker Green DND-26 and MitoTracker
Green were obtained from Thermo Fisher Scientific (Waltham, MA). The
CellTiter 96 Aqueous One Solution (MTS) Cell Proliferation Assay Kit
was from Promega (Madison, WI). The study utilized Human Pancreatic
Cancer cells (MIA PaCa-2) sourced from the American Type Culture Collection
(ATCC-CRL-1420). These cells were maintained in DMEM medium, which
was supplemented with 10% FBS, 4 mM l-glutamine, 1 mM sodium
pyruvate, and 1% penicillin/streptomycin (all acquired from Sigma),
and cultured at 37 °C in 5% CO₂. The viability of
the cells was routinely assessed using the Trypan Blue exclusion test
on a Bio-Rad TC20 automated cell counter. Upon reaching approximately
95% confluence, the cells were harvested by treating them with 0.25%
trypsin-EDTA for subsequent experiments.

This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Institution (Hospital das Clínicas, Faculdade de Medicina, University of São Paulo, Brazil), CAPPesq, numbers 620/11 and 0410/09, and HCFMUSP 8/41. Written informed consent was obtained from all subjects before they participated in this study.
MSCs from amniotic fluid (AF) and bone marrow (BM) were obtained as described [12 (link),15 (link),18 (link),21 (link),22 (link)]. Briefly, amniotic fluid was collected from nine healthy patients at 15 to 18 weeks of gestation via amniocentesis for fetal karyotype diagnosis at the Obstetrics Service of the same institution. AF samples (2–5 mL) were centrifuged at 450× g for 10 min at room temperature, and the resulting cells were washed with PBS. Cells were cultured in minimal essential medium-α modification (α-MEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% heat-inactivated FBS (Vitrocell, Waldkirch, Germany) and 1% penicillin/streptomycin antibiotics (100 UI/mL and 100 µg/mL, respectively; Sigma Aldrich, Saint Louis, MO, USA). The cells were maintained in a 37 °C incubator with a humidified atmosphere of 5% CO₂, with the culture medium changed every 3–4 days for approximately 12 days, when the first colonies appeared. After the 4th to 6th passages, cells were frozen under liquid nitrogen.
Bone marrow samples were obtained via iliac crest aspiration from six healthy donors undergoing bone surgery in the Orthopedics Department. BM samples (2–10 mL) were diluted 1:2 (v:v) in PBS and centrifuged for 7 min at 700× g at room temperature and washed with PBS. Mononuclear cells were isolated using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Chicago, IL, USA), washed, plated in 75-cm² culture flasks (Santa Cruz Biotechnology, Dallas, TX, USA), and cultivated in an MSC medium. The MSC medium consisted of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% heat-inactivated FBS and 1% penicillin/streptomycin antibiotics, as described above. After transferring to flasks, the cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂, with the culture medium changed every 3–4 days. Before reaching confluence, cells were detached using a trypsin-EDTA solution (Gibco, Waltham, MA, USA) and seeded at a density of 5 × 10³ cells/cm². After the 4th to 6th passages, cells were frozen under liquid nitrogen.
BMMSCs and AFMSCs were kept frozen under liquid nitrogen until use. After thawing, cells from the second and third passages were used for the experiments.