Nb1rgb

NB1RGB (human skin fibroblast) (Riken BRC) and IMR90 (human lung fibroblast) (JCRB Cell Bank) cells were maintained in MEMα (Wako, Tokyo, Japna) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cell cultures were passaged (1:4, every 3 days) and maintained at 37°C under 5% CO₂. We defined two types of each cell line according to the number of days in culture: NB1RGB: young cells: from 8 to 20 days and aging cells: from 60 to 70 days; IMR90: young cells: from 6 to 12 days and aging cells: from 30 to 40 days³⁴ (link)
NB1RGB cells were transiently transfected using lipofection (Lipofectamine 3000, Invitrogen, Carlsbad, CA, USA) with plasmids expressing SOD2 (pcDNA6.2-SOD2, cloned from pENTR221-SOD2 (DNAFORM ID 100008212) using Gateway cloning system (Invitrogen, Carlsbad, CA, USA).)

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.