Penicillin streptomycin amphotericin b

Ten cell lines from different organs and animal species were chosen in this study (Table 1). Cell culture medium for HEK-293T, MDCK, Paki, Pabr, Palu, and PK15 cells was Gibco™ Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher) with 10% FBS (Biological Industries), 1.5 g NaHCO₃, 1 mM sodium pyruvate (Thermo Fisher), and 1× penicillin–streptomycin–amphotericin B solution (Biological Industries). Cell culture medium for Vero, Caco-2, and Fcwf-4 cells was Minimum Essential Medium Eagle (MEM) (Sigma-Aldrich, St. Louis, MO, USA) with 10% FBS, 1.5 g NaHCO₃, 1 mM sodium pyruvate, 0.01% NEAA (Thermo Fisher), and 1× penicillin–streptomycin–amphotericin B solution. Cell culture medium for IEC-6 was DMEM with 5% FBS, 0.1 U/mL 95% bovine insulin (Sigma-Aldrich), 1.5g NaHCO₃, 1 mM sodium pyruvate, and 1× penicillin–streptomycin–amphotericin B solution. Eleven cells were divided into four groups according to their growth rate. HEK-293T, Paki, PK15, and MDCK cells belonged to the first group and grew to 70% confluence for one day. Vero, Pabr, Caco-2, and IEC-6 cells belonged to the second group and grew to 70% confluence for two days. Palu and Fcwf-4 cells belonged to the third group and grew to 70% confluence for three days. MFK cells needed four days to grow to 70% confluence and belonged to the fourth group.

All cell handling procedures described herein were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments and were approved by the Local Bioethics Committee of Wroclaw Medical University (registry number KB-177/2014). Written, informed consent for using tissue samples for research purposes was obtained from each and every patient prior to surgery.
Human mesenchymal stem/stromal cells were isolated from subcutaneous adipose tissue. Patients' fat biopsies (patient age ranged from 22 to 77 years) were obtained during total hip/knee arthroplasty or other open procedures connected with fracture reduction and fixation. Samples were placed in Hanks' balanced salt solution (HBSS) (Sigma-Aldrich, Germany) and transferred to the laboratory. According to a standard protocol [32 (link)], tissue samples were washed two times in HBSS with 1% antibiotic solution (Penicillin/Streptomycin/Amphotericin b, Sigma-Aldrich, Germany) and separated using surgical scissors. The minced tissue was digested in collagenase type I solution (1 mg/mL, Sigma-Aldrich, Germany) for 40 minutes at 37°C. After centrifugation (1200 ×g, 10 min) the supernatant was discarded and the pellet containing the cells was resuspended in culture medium and placed in a cell culture flask.

Human subcutaneous adipose tissue was obtained from patients undergoing elective lipoaspiration with informed consent (The University of Texas MD Anderson Cancer Center Institutional Review Board registrations IRB00001035, IRB00003657, IRB00004920, and IRB00006075). Adipose tissue was washed thoroughly, minced, and incubated with Ringers lactate containing a combination of collagenase I and II and a neutral protease (Matrase ^TM Reagent, InGeneron Inc. Houston TX) in a Tissue Processing Unit (Transpose RT^TM System, InGeneron Inc. Houston TX) for 30 minutes at 40°C. Subsequently, the cell suspension was filtered through a 100-μm filter, washed twice, and then centrifuged at 600 rpm for 5 minutes. The adipose stromal vascular fraction was resuspended in αMEM with 20% FBS, L-glutamine, and penicillin-streptomycin-amphotericin B (Sigma-Aldrich) at 37°C in 5% CO₂. Red blood cells in the supernatant and nonadherent cells were removed after 48 hours. For all experiments shown, human subcutaneous adipose tissue-derived cells were used prior to passage 6.

Collected amniotic fluid, at a 0.1–5 mL volume, was transferred from syringes to tubes under sterile conditions of a laminar chamber. The content of the syringes was inspected macroscopically for contamination, such as residual blood. Then, the tubes were centrifuged at 350×g for 10 min, the supernatant was discarded, and the precipitate was suspended in Dulbecco’s Modified Essential Medium (DMEM/Ham’s F12, Sigma, Germany) supplemented with 20% fetal bovine serum (FBS, Sigma, Germany), 10 ng bFGF (Sigma, Germany), and 1% antibiotic solution (penicillin/streptomycin, amphotericin B, Sigma, Germany). The viability of the cells was determined by the trypan blue test. Then, the cells were transferred to 35 mm Petri dishes. The dishes were incubated at 37°C with 5% CO₂ and constant humidity. The culture medium was replaced every 48 h. The cells were cultured until they reached confluence and then transferred to new Petri dishes.

To determine the cytotoxic potential of the selected compounds, the human epithelial breast cancer cell line MDA-MB-231 (American Type Culture Collection (ATCC), Manassas, VA, USA) was used. The cells were cultured in DMEM (Dulbecco’s modified essential media) (cat. No. 30-2002, ATCC, Manassas, VA, USA) with 10% fetal bovine serum (FBS) (cat. No. F7524, Sigma-Aldrich Co. LLC, St. Louis, MO, USA), 1 mM L-glutamine (cat. No. G7513, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) and penicillin/streptomycin/amphotericin B (100 units/mL: 10 mg/mL: 25 μg/mL) (cat. No. A5955, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at 37 °C, 5% CO₂ and optimal humidity.