[19–21], or by using fibroblasts from skin biopsies derived from donors with increasing age [22]. The observed age-related changes in fibroblasts include cell morphology, metabolism [19], reduced proliferative potential [19,22,23], loss of responsiveness to growth factors [24], decline in the production of extracellular matrix proteins such as type I and III collagens [25], and overexpression of proteases involved in the degradation of the extracellular matrix [26]. Reduction of telomere length [27], accumulation of free radical-induced DNA damages, and decrease of DNA repair [28], are molecular mechanisms associated with and promoting senescence. The above described modifications of fibroblast properties observed throughout serial in vitro expansion may all more or less participate to the in vivo age-related changes of the skin. A wide diversity of characteristics have been reported for fibroblasts derived from different tissues [29]. Even in a single tissue, heterogeneity is an important intrinsic property of fibroblasts residing at different micro anatomical localizations, which has to be taken into consideration when studying cellular aging. In human skin, it has been reported that cultured fibroblasts isolated from the upper and the deeper dermis, at the same anatomic site, exhibit distinct properties in regard to cellular morphology and proliferative potential [30,31], elaboration of the extracellular matrix [32–34], the production [35] and response [36,37] to growth factors and cytokines. However, little is currently known concerning the specific functions of these different fibroblast subpopulations, and their respective modifica- tions and fate during the process of skin aging. The aim of the present work was to investigate the age-related modifications of distinct human dermal fibroblast populations and their possible implication in skin aging. Fibroblasts were obtained from the superficial layer of the dermis, named ‘‘papillary dermis’’, and from the deeper dermal layer, named ‘‘reticular dermis’’. Matched pairs of papillary (Fp) and reticular fibroblasts (Fr), derived from donors of different ages were studied and analyzed for cellular morphology, secretion of growth factors and cytokines, production of matrix proteases, growth characteristics, and their capacity to promote epidermal morphogenesis. Materials and Methods Tissue and cell sample preparation Samples of human mammary skin (sun-protected skin areas) were obtained from adult Caucasian women, after plastic surgery. Informed consent was obtained before tissue collection, according to European guidelines. Age of donors ranged from 19 to 74 years. After removing the subcutaneous tissue, skin samples were sliced according to depth using a dermatome. The upper part of skin samples (from the surface to a 0.3 mm depth) contained the epidermis and the papillary dermis. The deeper part of skin samples (depth .0.7 mm) corresponded to the reticular dermis. Quality of full skin samples and sections was monitored by histological analysis. Collagen fibers were stained with Sirius red (Interchim, Montluc¸on, France). The epidermis was separated from papillary dermis by dissection, after treatment with 0.25% trypsin for 1.5 hours at 37uC (Invitrogen, Carlsbad, CA). For the preparation of papillary (Fp) and reticular fibroblasts (Fr), pieces of deep and superficial dermis were placed in culture in MEM supplemented with 10% fetal bovine serum (FBS) (D- Dutscher, Brumath, France), penicillin-streptomycin (20 U/ml) (Biochrom, Cambridge Ldt, UK), and glutamine (2 mM) (Invitro- gen, Carlsbad, CA), and maintained at 37uC in a 90% humidified atmosphere containing 5% CO2. After migration of fibroblasts outward dermal explants, these cells were amplified in culture and Fp and Fr in Skin Aging stored in liquid nitrogen as frozen aliquots until use. Match pairs of Fp and Fr were used for the different investigations at the same early passage. The keratinocytes used in this study were from the epidermis of a single skin sample from a 28 years old donor. Epidermal pieces were dissociated by incubation in 0.25% trypsin for 1.5 hours at 37uC (Invitrogen, Carlsbad, CA). Keratinocytes were plated on a feeder-layer of growth-arrested 3T3 murine fibroblasts, and amplified according to the method described by Rheinwald and Green [38]. Keratinocytes were then frozen in liquid nitrogen until use for three dimensional skin reconstruction experiments. Quantification of secreted cytokines, MMPs and TIMPs Samples of Fp and Fr were plated at a density of 16104 cells/ cm2 in duplicate 60 mm culture dishes in a medium composed of MEM supplemented with 10% fetal bovine serum (FBS) (D- Dutscher, Brumath, France), penicillin-streptomycin (20 U/ml) (Biochrom, Cambridge Ldt, UK), and glutamine (2 mM) (Invitro- gen, Carlsbad, CA). Culture medium was renewed every 3 days after fibroblast plating. To minimize the quantities of endogenous cytokines, growth factors, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) present in the medium, cultures were maintained in the presence of a low serum concentration (1%) the 3 last days before the analysis of secretion profiles of Fp and Fr (from day 6 to day 9). At day 9, supernatants from Fp and Fr cultures were sampled and processed for the quantification of cytokines, MMPs, and TIMPs by ELISA: interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP- 1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), MMP-2, MMP-3, TIMP-1 and TIMP-2 (Human Quantikine kits, R&D Systems, Minneapolis, MN), MMP-1 (Biotrack kit, Amersham Biosciences, Buckinghamshire, UK). Background concentrations of cytokines, MMPs and TIMPs present in the culture medium containing 1% FCS in the absence of cells were found very low, often below the detection threshold of quantification assays. When detectable, these values were deduced from concentrations measured in cell supernatants. Values were then normalized according to cell numbers counted in the respective cultures and expressed in pg/ml (cytokines) and ng/ ml (MMPs and TIMPs) per 16105 cells. Flow cytometry analysis Morphological characteristics of young and old Fp and Fr were analyzed by flow cytometry (FACS Aria, BD Biosciences, San Jose, CA). Cultures of Fp and Fr from 3 matched pairs of young and 3 of old donors were trypsinized 7 days after plating, and re- suspended in PBS supplemented with 0.2% bovine serum albumin (BSA) at 46105 cells/ml. The respective cell samples were analyzed for size (forward-scatter, FSC) and granularity (side- scatter, SSC). Results were referred to viable cells, discriminated from dead cells by 7 AAD staining (eBioscience, San Diego, CA). Short term growth Previously amplified fibroblasts in primary culture were plated in triplicates into 10 cm2 wells at a density of 16104 cells/cm2, and cultured in a medium composed of MEM supplemented with 10% FBS (PAN Biotech GmbH, Aidenbach, Germany), penicillin- streptomycin (20 U/ml) (Biochrom, Cambridge Ldt, UK), and glutamine (2 mM) (Invitrogen, Carlsbad, CA). Cell counts were performed at days 7, 14, and 21 after culture initiation. Cellular expansion values corresponded to numbers of cells counted after 7, 14 and 21 days of culture divided by the number of cells initially plated. PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e4066