Each eluate sample diluted 2-folds with DMEM containing 10% FBS was used for as an eluate sample without fibroblasts. on diabetic mice, the dry sheet-treatment groups using autologous or allogeneic cells showed significantly accelerated wound closure compared with that in the no-treatment group. The storage stability of the dry sheet was better at refrigeration temperature than at room temperature and remained stable for at least 4?weeks. Our data indicated that allogeneic dry sheets represent a promising new tool for regenerative medicine that promotes wound healing. for 5?min at 4?C, and supernatants were Phentolamine HCl collected and stored at???30?C until measurement. The concentrations of VEGF, HGF, FGF-2, and HMGB1 in the supernatant were measured using Quantikine Immunoassay Kits (R&D Systems) and HMGB1 ELISA Kit Exp (SHINO-TEST CORPORATION, Kanagawa, Japan), respectively, according to Phentolamine HCl the manufacturers instructions. Cell proliferation, VEGF and HGF production analysis in fibroblasts following incubation with eluate from cell sheets The eluate samples were prepared by immersing each cell sheet in 200 L DMEM (Thermo Fisher Scientific) without FBS for 24?h under normoxic conditions. The supernatant was collected after centrifugation (2460 em g /em , 4?C, 5?min). Fibroblasts were seeded in 96-well plates at 8000 cells/well in 100 L DMEM with 10% FBS, followed by addition of 100 L of either the eluate sample or DMEM without FBS. Each eluate sample diluted 2-folds with DMEM containing 10% FBS was used for as an eluate sample without fibroblasts. After 48?h of incubation under normoxic conditions, the culture supernatant was collected for the measurement of VEGF and HGF, followed by the cell proliferation assay. DMEM (100 L) with 5% FBS containing 10% WST-8 reagent (Cell Count Reagent SF) was added to each well and incubated for 1?h under normoxic conditions. The absorbance of the supernatant was measured at 450?nm. The cell proliferation rate was calculated using the fibroblasts cultured in DMEM as the control. The concentrations of VEGF and HGF in the culture supernatant were measured by ELISA, and the production ratios were calculated by following formula: [(supernatant of fibroblasts cultured with the eluate sample)???(supernatant of the eluate sample without fibroblasts)]/(supernatant of fibroblasts cultured with DMEM). Three independent experiments were performed in triplicate. Neutralizing antibody experiment Dry sheet eluate samples were prepared by the same procedure as for the cell proliferation analysis described above. DMEM containing rFGF-2 (0 or 5?ng/mL, FUJIFILM Wako) were also prepared. These samples were incubated for 60?min at 37?C with the anti-FGF-2 neutralizing antibody (30.3?g/mL, #05-117, Phentolamine HCl clone bFM-1, Merck Millipore, Darmstadt, Germany), or the control antibody (Mouse IgG1 isotype control, clone11711; 30.3?g/mL, MBA002, R&D Systems). Fibroblasts were seeded in 96-well plates at 8000 cells per well in 100 L DMEM with 1% FBS, followed by the addition of 100 L of either the eluate sample with the antibodies, or DMEM containing rFGF-2 (0 or 5?ng/mL) with antibodies. After a 48?h incubation under normoxic conditions, the culture supernatant was aspirated and the cell proliferation assay was performed. DMEM (100 L) with 0.5% FBS containing 10% Cell Count Reagent SF was added to each well and incubated for 2?h under normoxic conditions. The absorbance of the supernatant was measured at 450?nm. The cell proliferation rate was calculated using fibroblasts cultured in 0?ng/mL rFGF-2 with the control antibody as the control. Three independent experiments were performed in triplicate. Mouse cutaneous ulcer model and cell sheet transplantation Male C57BL/6N mice were intraperitoneally administered streptozotocin (STZ; FUJIFILM Wako) at 55?mg/kg every 24?h for five consecutive days. Mice with blood glucose values of??300?mg/dL on days 9 and 10 after administration of STZ were classified as diabetic mice. A skin ulcer was prepared in the diabetic mice on the 14th day after the last administration of STZ. Male C57BL/6N mice were anaesthetized with 2% isoflurane via inhalation, and a 6?mm full-thickness skin defect was created on the dorsal skin using a biopsy punch (n?=?6 per group). Living sheets and FT sheets were transferred onto the skin defect using a 1000 L wide-bore tip, and dry sheets were handled using tweezers. Each cell sheet was prepared from male C57BL/6N mice (autologous) and male C3H/He mice (allogeneic). All wounds were covered with ADAPTIC (#2012; Acelity, San Antonio, TX, USA) and Derma-aid? (ALCARE, Tokyo, Japan) and fixed with Silkytex bandage (#11893; ALCARE) for the first 24?h. On the first day, all wounds were Rabbit Polyclonal to PKCB1 covered with Airwall Fuwari (# MA-E050-FT; Kyowa, Osaka, Japan) and fixed using Silkytex bandage9. Each wound was photographed using a.
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