human neonatal foreskin fibroblasts (hdfs)  (Lonza)

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of Polymeric Particles for Modular Tissue Cultures in Developmental Engineering

doi: 10.3390/ijms24065234

Figure Lengend Snippet: ( I , II ) Micrographs of human dermal fibroblasts (HDFs) cultured on spherical modular scaffolds. Aliquots of 1 mL HDFs (10 3 cells/mL) were seeded in each well of 24-well tissue culture plate containing 5 mg poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA) or polystyrene (PS) particles (diameter: 5, 10, 20, 30 or 100 µm) submerged in 1 mL DMEM and cultured for 14–30 days. The phase contrast micrographs of HDFs ( I ) cultured on ( A – C ) PMMA, ( D – F ) PLA and ( G – I ) PS particles; the fluorescent micrographs of HDFs ( I ) cultured on ( J – L ) PMMA, ( M – O ) PLA and ( P – R ) PS particles were captured. Blue: nuclei stained by DAPI, Green: cytoskeleton stained by phalloidin. (scale bar = 100 µm). Scanning electron micrographs of HDFs ( II ) cultured on ( A – F ) PMMA, ( G – L ) PLA and ( M – R ) PS particles were captured. Cell colonization strategies via cell bridging and cell stacking are highlighted via White and Black arrows respectively. (scale bar = 10 µm except G = 1 µm).

Article Snippet: The medium used for the culture of Neonatal foreskin human dermal fibroblasts (HDFs, Intercytex, Manchester, UK) was Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Walkersville, MD, USA) containing 4.5 g/L glucose supplemented with 2 mM L-glutamine (Gibco, Paisley, UK), 100 IU/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, New York, NY, USA), and 10% ( v / v ) fetal bovine serum (FBS, Gibco, Paisley, UK).

Techniques: Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

doi: 10.3390/ijms21031022

Figure Lengend Snippet: EV secretion was increased in replicative senescent dermal fibroblasts. ( A ) Senescence-associated (SA)-β-gal assay in young HDFs (PDL < 10) and senescent HDFs (PDL > 50). Scale bar = 50 μm. Data are means ± SD of three independent experiments on two independent senescent cell lines (*** p < 0.001). ( B ) Western blot analysis of p21 and p16 in young versus senescent HDFs. GAPDH was the loading control. ( C ) Levels of EVs derived from equal numbers of young and senescent HDFs. Protein concentrations in isolated EVs were determined by BCA assay. Data are means ± SD of four independent experiments using two independent senescent cell lines (*** p < 0.001). ( D ) Dynamic light scattering analysis of EVs derived from young and senescent HDFs. ( E ) Western blot analyses of EV markers. Five micrograms of EV protein were subjected to immunoblot analysis with anti-CD81, anti-CD9, anti-Alix, and anti-HSP90. GAPDH was the loading control.

Article Snippet: Human dermal fibroblasts derived from the skin of a 33-year old adult (HDFs) or neonatal foreskin (HDFs-Neo) were purchased (Lonza, Basel, Switzerland) and cultured in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland).

Techniques: Western Blot, Derivative Assay, Isolation, BIA-KA

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

doi: 10.3390/ijms21031022

Figure Lengend Snippet: EV biogenesis increased in senescent dermal fibroblasts. ( A ) Schematic image of sandwich ELISA using anti-CD81 antibody to detect EVs. ( B ) Conditioned media used to culture equal numbers of young and senescent HDFs were harvested at the indicated time points. EV levels were quantitated by sandwich ELISA for CD81. Data are means ± SD of three independent experiments using a senescent cell line (*** p < 0.001). ( C ) HDFs were labeled with a fluorescent lipid molecule N-Rh-PE (red) for the indicated time periods and co-stained with anti-CD63 antibody (green). Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI; blue). Fluorescence images were taken under a confocal microscope. Representative images are shown. Scale bar = 20 μm.

Article Snippet: Human dermal fibroblasts derived from the skin of a 33-year old adult (HDFs) or neonatal foreskin (HDFs-Neo) were purchased (Lonza, Basel, Switzerland) and cultured in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland).

Techniques: Sandwich ELISA, Labeling, Staining, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

doi: 10.3390/ijms21031022

Figure Lengend Snippet: Senescent HDF-derived EVs attenuate the dermal effect on keratinocyte differentiation but evoke proinflammatory cytokine IL-6. Human epidermal keratinocytes (HEKs) in culture were treated with EVs derived from 5 × 10 5 young and senescent HDFs for 4 days. HEKs cultured without EV treatment for 4 d served as the control (-). Cells were analyzed for mRNA ( A ) and protein ( B ) expression of keratinocyte differentiation-related (KRT1, LOR) or barrier function-related (BLMH) markers by qRT-PCR and western blot analyses, respectively. The mRNA levels were normalized to those of RPL13A. GAPDH was the loading control for western blot analysis. KRT1, keratin 1; LOR, loricrin; BLMH, bleomycin hydrolase. ( C , D ) HEKs were treated with EVs derived from 5 × 10 5 young and senescent HDFs. Conditioned media at 24 h and 4 d post-treatment with EVs were harvested for the determination of secreted IL-8 ( C ) and IL-6 ( D ) levels, respectively, with specific ELISA kits. Data (in A , C , D ) are means ± SD of three independent experiments using two senescent cell lines (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant).

Article Snippet: Human dermal fibroblasts derived from the skin of a 33-year old adult (HDFs) or neonatal foreskin (HDFs-Neo) were purchased (Lonza, Basel, Switzerland) and cultured in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland).

Techniques: Derivative Assay, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay