Colony assays Fibroblasts from the different individual samples were plated at the low density of 100 cells per 100 mm diameter culture dish (Corning, New York, NY). Cultures were performed for 12 days in a medium composed of MEM supplemented with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO), penicillin streptomycin (20 U/ml) (Biochrom, Cambridge Ldt, UK), and glutamine (2 mM) (Invitrogen, Carlsbad, CA), and maintained at 37uC in a 90% humidified atmosphere containing 5% CO2. Medium was completely renewed every 4 days. Cultures were fixed in ethanol (70%) and stained with Eosin (RAL, Bordeaux, France) and Methylen Blue (RAL, Bordeaux, France). Colonies were counted and photographed under an inverted microscope coupled with a CCD camera (DMIL, Leica Microsystems, Wetzlar, Germany). The area occupied by cells in individual colonies was scored using Image J software (NIH, Bethesda, MA). Colony counts were based on the analysis of triplicate dishes. Colonies were classified according to the surface occupied by cells and separated into 3 2 groups: large colonies (.1.5 mm ), colonies of intermediate size 2 2 (0.5–1.5 mm ), and small colonies (,0.5 mm ). Correspondence between the surface of colonies and cell numbers was determined by cell counting on micro-photographs of fixed colonies. Three-dimensional skin reconstruction and xenograft experiments Reconstructed skins were prepared as previously described [39]. Briefly, fibroblasts (16106 cells per sample of reconstructed dermis) were embedded into a bovine type I collagen gel (Symate`se, Chaponost, France). Diameters of each dermal equivalent (lattice) were measured using a millimeter scale during the 4 days of lattice contraction. Thereafter, keratinocytes (56104 cells per sample of reconstructed epidermis) were seeded onto the lattices, stuck to the bottom of 60 mm diameter Petri dishes. Cultures were maintained for 1 week immersed in a medium composed of MEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Sigma, St Louis, MO), Epidermal Growth Factor (EGF) (10 ng/ml) (BD Biosciences, San Jose, CA), Hydrocortisone (0.4 mg/ml) (Sigma, St Louis, MO), and Cholera Toxin (0.1 nM) (Biomol Int., Plymouth, PA). Complete epidermal stratification and full differentiation was obtained 1 week after raising the system at the air-liquid interface. During the whole process of skin reconstruction, cultures were maintained at 37uC, in a fully humidified atmosphere containing 5% CO2. Transplantation of human reconstructed skin samples onto immunodeficient athymic mice were performed using a previously published procedure [40]. Experiments were conducted in agreement with the European guidelines (European instruction 86/609), and were approved by an animal care and use committee. Recipient mice were 6-week-old ICO Swiss nu/nu mice. Bandages were maintained at least 1 week after grafting. Human reconstructed skin samples were harvested 5 weeks after grafting, and processed for histological analysis. Histology Samples of human and reconstructed skin were fixed in paraformaldehyde, dehydrated, and embedded in paraffin. Histological examinations of tissue sections were performed after hematoxylin-eosin-saffron (HES) staining, using a Leica DMR light microscope (Leica Microsystems, Wetzlar, Germany). Thicknesses of spinous, granular and cornified layers, as well as total nucleated cell layers, were measured by image analysis using the image J software (16 different quantifications were performed for each experimental condition). Fp and Fr in Skin Aging Immunofluorescence Samples of reconstructed skin were embedded in Tissue-Tek (Miles Inc., Elkart, IN) and frozen in liquid nitrogen, and cut into 5 mm thickness sections (cryostat, CM3050 S, Leica, Microsys- tems, Wetzlar, Germany). Filaggrin was stained using a mouse monoclonal antibody (clone BT-576, Biomedical technologies Inc., Stoughton, MA), revealed using a FITC-conjugated rabbit anti-mouse polyclonal antibody (DAKO DK-2600, Glostrup, Denmark). Nuclei were stained using propidium iodide (Sigma, St Louis, MO). Stained tissue section were observed and imaged under fluorescence microscope (DMR, Leica, Microsystems, Wetzlar, Germany). Cell samples were plated on glass slides in micro-culture chambers (Lab-Tek, Nalgen Nunc international, NY), and cultured for 3 days in MEM / 10% FBS. Microphotographs of cultured cells were taken under light microscopy (DMIL, Leica Microsystems, Wetzlar, Germany). For immunofluorescence labeling, cells were fixed in 4% paraformaldehyde (Carlo erba, Rodano, Ilaly), washed in TBS, and permeabilized in TBS-Tween 0.5% (Sigma, St Louis, MO). Vinculin was stained using a mouse FITC-conjugated monoclonal antibody (Clone hVIN-1, Sigma, St Louis, MO). Actin filaments were stained using TRITC- conjugated phalloidin (Invitrogen, Calsbad, CA). Stained cultured cells were observed and imaged under fluorescence microscope (DMIRB, Leica, Microsystems, Wetzlar, Germany). Statistics Correlation between concentrations of secreted cytokines, MMPs, TIMPs and donor age were estimated by linear regression. Correlation coefficient (R) and statistical significance (P) were considered acceptable when values were R.0.5 and P,0.05, respectively. For the other studies, differences between experi- mental groups were analyzed using the ANOVA mixed model. Differences were considered as statistically significant when P values were ,0.05. When necessary, data were transformed in logarithm to satisfy conditions of normality and homogeneity. When variances were unequal, ANOVA was run on rank- transformed data. Results Experimental definition of Fp and Fr cells The process used to generate Fp and Fr samples is schematized in Fig. 1A and 1B. Fp and Fr were respectively isolated from superficial dermis (between the surface to 0.3 mm depth) and deep dermis (more than 0.7 mm depth). Cutting at a depth of 0.3 mm allowed the recovery of superficial papillary dermis with the epidermis (Fig. 1C). The skin part that was discarded after cutting at 0.7 mm depth to select the deep reticular dermis is visualized in Fig. 1E. After few days of culture, Fp were characterized by a spindle-shaped morphology (Fig. 1F), although cultured Fr had a stellate morphology (Fig. 1G). Staining of actin indicated a diffuse organization of cytoskeleton in Fp (Fig. 1H). On the contrary, actin filaments were marked in Fr (Fig. 1I). Focal adhesion points, revealed by vinculin staining, were more abundant in Fr (Fig. 1I) than in Fp (Fig. 1H). Main histological characteristics of human young and old mammary skin The age-related characteristics of human skin are illustrated by the representative histological sections of mammary skin biopsies obtained from a young (19 yr) and an old (74 yr) adult donor (Fig. 2A and 2B, respectively). In general, the epidermis was thicker in young compared to old skin. The rete-ridge structures PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e4066