Penicillin and streptomycin

Neurospheres were chopped to 0.2 mm 3 days before plating to reach a defined size of 0.3 mm. Spheres were plated in five technical replicate wells/condition in 96-well plates (flat bottom, Greiner, # 655,180) with one sphere/well plated in 100 µL of differentiation medium containing 13 concentrations of CHMs: 0.000001, 0.00001, 0.0001, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.075, 0.10, 0.50, and 1.0 mg/mL LGT and 0.000001, 0.00001, 0.0001, 0.001, 0.0025, 0.005, 0.01, 0.1, 0.25, 0.50, 0.75, 1.0, and 2.0 mg/mL TM. The differentiation medium composition was DMEM (Thermo Fisher, #31,966,021), Hams F12 (Thermo Fisher, #31,765,027) 2:1 supplemented with 1% of N2 (Thermo Fisher, #17,502–048) and 1% penicillin and streptomycin (Pan-Biotech, #P06-07,100). Neurospheres were exposed for 3 (for migration/adhesion endpoints) or 5 (for differentiation endpoints) days and all experiments were performed under standard culture conditions at 37 °C with 5% CO₂. For the latter, on day 3, half of the exposure/solvent medium was exchanged and the supernatant was used to detect cytotoxicity by measuring lactate dehydrogenase (LDH) leakage.

A well-established cell line was used for the evaluation of the cytotoxicity and the osteogenic potential in direct contact with the materials. Pre-osteoblastic MC3T3-E1 cells (DSMZ Braunschweig, Germany, ACC-210) from newborn mouse calvaria were used. These cells exhibit an osteoblastic phenotype and have the potential to differentiate into osteoblasts [44 (link)]. The cells were cultured in α-MEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) (PAN-Biotech, Aidenbach, Germany), 100 μg/mL penicillin and streptomycin (PAN-Biotech, Aidenbach, Germany), 2 mM L-glutamine (PAN-Biotech, Aidenbach, Germany), and 2.5 μg/mL amphotericin (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator at 37 °C, 5% CO₂ (Heal Force, Shanghai, China). The cell culture medium was replaced twice weekly and confluent cells were detached using trypsin-0.25% ethylenediaminetetraacetic acid (EDTA) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). MC3T3-E1 cells between passages P14-16 were used for the experiments.

293T (human embryonic kidney; ATCC CRL-3216), Vero E6 (African green monkey kidney; ATCC CRL-1586), LLC-MK2 (rhesus macaque kidney; ATCC CCL-7) and EpoNi/22.1 (Buettikofer’s epauletted fruit bat kidney, [44 (link)], kindly provided by M.A. Müller and C. Drosten) cell lines were obtained from collaborators. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech) supplemented with 10% fetal bovine serum (FBS; PAN Biotech) as well as 1x penicillin and streptomycin from a 100x stock solution (PAN Biotech). The cell lines were cultivated at 37 °C and 5% CO₂ in humidified atmosphere. Transfection of 293T cells was achieved by calcium phosphate precipitation.

MC3T3-E1 pre-osteoblastic cells (DSMZ, Braunschweig, Germany, ACC-210) isolated from newborn mouse calvaria have the capacity to differentiate into osteoblasts in vitro [56 (link)], and have been used for the investigation of cell adhesion, viability, proliferation, and differentiation of biomaterials. The pre-osteoblastic cells were cultured in complete medium comprising alpha-MEM cell culture medium (PAN-Biotech, Aidenbach, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS) (PAN-Biotech, Aidenbach, Germany), 100 μg/mL penicillin and streptomycin (PAN-Biotech, Aidenbach, Germany), 2 mM L-glutamine (PAN-Biotech, Aidenbach, Germany), and 2.5 μg/mL amphotericin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator at 37 °C, 5% CO₂ (Heal Force, Shanghai, China). The culture medium was replaced twice weekly. The cells were detached using trypsin-0.25% ethylenediaminetetraacetic acid (EDTA) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Passages 12–17 of pre-osteoblastic cells were used for the viability and differentiation experiments.

The CMGC cell line CIP established by N. Sasaki was used in this study [36 (link)]. The validated CIP cell lines obtained from N. Sasaki were delivered from an anonymous veterinary pharmacology laboratory. CIPp was derived from primary CMGCs, and CIPm was derived from metastatic regional lymph nodes. CIPp and CIPm cells were cultured in the Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin and streptomycin (PAN-Biotech, Aidenbach, Germany). The cultured cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂, and the medium was replaced every 2–3 days.