Penicillin streptomycin fungizone

The human keratinocyte cell line (HaCaT) was obtained from Cell Line Services (Oppenheim, Denmark) and were cultured, as well as, the mouse fibroblast cells, L929 (NCTC) (ECACC 85103115), at 37 °C in a humidified atmosphere of 95% air and 5% CO₂, as monolayers using Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine without pyruvate (Lonza, Verviers, Belgium) containing 10% (v/v) fetal bovine serum (FBS, Biowest, Nuaillé, France) and 1% (v/v) Penicillin-Streptomycin-Fungizone (Lonza, Verviers, Belgium).

For five of the 14 myopathy patients, we could obtain skin biopsies to be subcultured as fibroblast cell lines. Five fibroblast cell lines obtained from nonmyopathy individuals were included as controls, together with the commercial human dermal fibroblast (Gibco™ C0045C) lines HDFa16 and HDFn16. Fibroblast cell lines were cultured in Ham’s F10 + l-Glutamine medium (Thermo Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (Lonza, Basel, Switzerland) and 1% penicillin/streptomycin/fungizone (Lonza) at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were trypsinized at 80–90% confluency and cultured with fresh medium in a 25 cm² culture flask at 10⁵ cells in 5 mL at seeding. Subculture took place in a 175 cm² culture flask. At 80–90% confluency, cells were harvested and washed twice in ice-cold PBS. The cells were pelleted at 800× g at 4 °C, and stored at −80 °C for further analysis.

The cytocompatibility of the ABVF putty was assessed via a standard alamarBlue assay, according to the manufacturer’s protocol. Briefly, 10,000 MG-63 osteoblast cells (ATCC, Manassas, VA, USA) were seeded in each well of a 96-well plate. Cells were grown in Dulbecco’s Modified Eagle Media (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-fungizone (Lonza, Walkersville, MD, USA). Cells were incubated at 37 °C and 5% CO₂. After reaching 60% confluence, 100 μL of release media from ABVF was added to each well. Control wells received 1X PBS only. After 48 h of incubation, cells were washed with 1X PBS three times. Fresh media was added to the wells followed by addition of alamarBlue to a 10% final concentration. Subsequently, wells were incubated at 37 °C for 4 h. After the incubation, absorbance was read at 570 nm and 600 nm (Spectramax m5, Molecular Devices, Downingtown, PA, USA). Cell viability was calculated using the following equation:
where O1 = molar extinction coefficient (E) of oxidized alamarBlue (Blue) at 570 nm, O2 = E of oxidized alamarBlue at 600 nm, A1 = absorbance of test wells at 570 nm, A2 = absorbance of test wells at 600 nm, P1 = absorbance of positive growth control well (cells plus alamarBlue but no test agent) at 570 nm, P2 = absorbance of positive growth control well (cells plus alamarBlue but no test agent at 600 nm).