Amphotericin b

Spheroids from oral mucosa-, skin-, and CBDC-derived cells were transferred into a new regular culture dish. After these cultures reached 50–60% confluence, the medium was changed to neurogenic induction medium. The medium used for neural cell differentiation was αMEM supplemented with l-glutamine, phenol red (Wako), 50 ng/ml nerve growth factor, 50 ng/ml brain-derived neurotrophic factor, 10 ng/ml NT-3 (all from Peprotech), 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Biological Industries). For Schwann cell differentiation, the medium used was αMEM supplemented with l-glutamine, phenol red (Wako), 5 μM forskolin (Sigma), 50 ng/ml heregulin-1β (Peprotech), 2% v/v N2 supplement (Invitrogen), 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Biological Industries). The cells were differentiated for 1 or 2 weeks, and 50% of the medium was changed every 2 days.

Human umbilical vascular endothelial cells (HUVECs), GBM8901 (BCRC Cat# 60164) (Chin. Med. J. (Taipei) 48: 177-184, 1991) glioblastoma and B16F10 (BCRC Cat# 60031) melanoma cell lines were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). HUVECs were maintained in M199 medium containing 10% FBS, 20 mM HEPES, 5 U/ml heparin, 100 U/ml of penicillin G, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B (Biological Industries, Cromwell, CT, U.S.A), and vascular endothelial cell growth supplement (ECGS) (Millipore, Billerica, MA, U.S.A.) in a humidified 37°C incubator. Other cells were maintained in DMEM (B16F10 cells) or RPMI1640 (GBM8901 cells) medium containing 10% FBS, 100 U/ml of penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Biological Industries, Cromwell, CT, U.S.A) in a humidified 37°C incubator. The murine LEC line SV-LEC was kindly provided by Dr. J.S. Alexander (Shreveport, LA). SV-LECs were cultured as previously described (34 (link), 35 (link))

Subcutaneous adipose tissue samples (approximately 500 g) were harvested from the hind leg area of Holstein Friesian cows (n = 5) at an abattoir (Haifa, Israel) during the winter season. Adipose samples were trimmed and washed with prewarmed phosphate-buffered saline (PBS, 38 °C) containing 100 µg/mL penicillin, 100 µg/mL streptomycin and 100 µg/mL Amphotericin B (Biological industries, Kibbutz Beit-Haemek, Israel). Each adipose sample was cut into 5 pieces (each about 50 g) and placed in prewarmed PBS (38 °C) containing 100 µg/mL penicillin, 100 µg/mL streptomycin and 100 µg/mL Amphotericin B (Biological industries, Israel) and transported to the cell culture laboratory within one hour after sampling. The study was in accordance with the regulations of Ministry of Health, Israel. Certificate Nu: #80. Adipose tissue was collected after cows were slaughtered by a certified worker at Haifa commercial slaughterhouse (32694 IL).