Antibiotic antimycotic

In this study, the effect of HHP combined with supercooling pretreatment on normal human skin fibroblasts (NB1RGB, Riken BRC, Tsukuba, Japan) was evaluated to validate a decellularization method using low-level HHP with supercooling pretreatment. As reported in our previous study [32 ], NB1RGB cells were cultured in alpha-modified Eagle minimum essential medium (MEM α, Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic–antimycotic (Nacalai Tesque, Kyoto, Japan) in a humidified CO₂ incubator (5% CO₂ at 37 °C). NB1RGB cells were suspended in MEM α culture medium without phenol red, supplemented with 10% FBS and 1% antibiotic–antimycotic to a concentration of 5.0 × 10⁵ cells/mL. The cell suspensions were subjected to supercooling pretreatment and HHP application. After treatment, the cell viability and proliferation were evaluated using a live/dead assay using calcein-AM and PI staining. Cell morphology was evaluated by scanning electron microscope (SEM).

In this study, the effect of HHP on normal human skin fibroblasts (NB1RGB, RIKEN BRC, Kyoto, Japan) was evaluated to validate a physical decellularization method using HHP. NB1RGB cells were maintained in alpha-modified Eagle minimum essential medium (MEM α, Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich, St. Louis, MO, USA) and 1% antibiotic–antimycotic (Nacalai Tesque, Kyoto, Japan) in a humidified CO₂ incubator (5% CO₂ at 37 °C). From a cryopreserved stock, NB1RGB cells were passaged twice before the HHP application experiments. Harvested NB1RGB cells were suspended in a culture medium of MEM α without phenol red supplemented with 10% FBS and 1% antibiotic–antimycotic to a concentration of 5.0 × 10⁵ cells/mL, and subjected to the HHP treatment. After treatment, cell morphology, cell viability, and cell proliferation were evaluated.