Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

Sumamry A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1×10⁻⁹ M), isobutyl methylxanthine (3.3×10⁻⁵ M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies. This research was supported by grant AG 01312 from the U.S. Public Health Service, Washington, D.C.     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process. (Mol Cell Biochem 269: 209–216, 2005)     

Comparative in vitro cytotoxicity of ethyl acrylate and tripropylene glycol diacrylate to normal human skin and lung cells

Summary The potential for occupational exposure to the esters of acrylic acid (acrylates) is considerable, and, thus, requires a greater understanding of the their toxicity. Confluent (70–90%) cultures of normal human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF), or bronchial epithelium (NHBE) were exposed to the monofunctional ethyl acrylate (EA), the multifunctional tripropylene glycol diacrylate (TPGDA), or TPGDA monomer in a radiation curable lacquer (Lacquer A) at equimolar dosages in order to determine human in vitro cytotoxicity. Viability of the cells after 2–24-h exposure to the representative monofunctional or multifunctional acrylate or solvent control was used to calculate an index of acute cytotoxicity (50% inhibitory dose; ID₅₀) and to determine the shape of the dose-response curves. TPGDA, Lacquer A, and EA were equally cytotoxic (ID₅₀≈0.1 μmol/cm²) to NHEK at equimolar doses. TPGDA or Lacquer A were more cytotoxic (≈100×) to NHDF or NHBE than EA. Sequential exposure of UV_A and TPGDA to NHEK indicate the potential for a synergistic cytotoxic response. These findings are consistent with observed decreases in free sulfhydryl groups (e.g., glutathione or cysteine) that parallel the dose-response-related decreases in viability. Together, these data suggest possible differences in toxicity between the monofunctional EA and multifunctional TPGDA to NHEK, NHDF, or NHBE, possibly due to the difference in the number of functional acrylate groups and/or physicochemical differences (e.g., vapor pressure) between the acrylates investigated.     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture

Summary During serial subcultures 50 μg per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 μg per ml) depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

New method for the isolation of endothelial cells from large vessels

Background aimsNumerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.MethodsWith the use of this method, the lumen of a bovine aorta was filled with wash medium and the outer surface of the sample was washed with alcohol for 30 seconds. Under a laminar flow hood, the inner surface of the sample was covered with filter paper. Collagenase type II was dripped onto the filter paper as a digestion enzyme. The digestion fluid was seeded in T25 flasks and fed with complete medium every 3 days.ResultsThe isolated cells were characterized by markers such as CD31, von Willebrand factor, 1,1′-dioctadecyl-1,3,3,3′,3′-tetramethylindocarbocyanine perchlorate acetylated low-density lipoprotein and angiogenesis behavior. The purity of endothelial cells was detected by flow cytometry to be of nearly 90% purity; these results were confirmed by immunostaining. Moreover, endothelial cells formed blood vessel–like tubes in a three-dimensional environment, which is specific dynamic behavior for endothelial cells.ConclusionsThe new strategy presented in the current report enables isolation of a highly pure population of endothelial cells that can survive long-term culture without inducing an overgrowth of fibroblast cells.     

尖顶羊肚菌胞外多糖提取物对皮肤成纤维细胞增殖和衰老的影响

不同浓度尖顶羊肚菌胞外多糖提取物作用人皮肤成纤维细胞（human skin fibroblasts,HSF）,检测对HSF细胞形态、细胞增殖、衰老相关β-半乳糖苷酶活性、羟脯氨酸含量的影响,探究尖顶羊肚菌胞外多糖提取物对HSF增殖和衰老的影响。结果显示,125μg/mL尖顶羊肚菌胞外多糖提取物使HSF细胞活力增加了25.2%,羟脯氨酸含量增加了12.1%,β-半乳糖苷酶活性降低了48.1%。说明适宜浓度尖顶羊肚菌胞外多糖提取物具有促进HSF细胞增殖、胶原蛋白合成,延缓细胞衰老的作用。     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.     

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes

Summary The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2–3 mm³) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700 U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1×10⁴ cells/cm². These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.     

Bisphosphonates induce the osteogenic gene expression in co‐cultured human endothelial and mesenchymal stem cells

Bisphosphonates (BPs) are known to affect bone homeostasis and also to have anti-angiogenic properties. Because of the intimate relationship between angiogenesis and osteogenesis, this study analysed the effects of Alendronate (AL) and Zoledronate (ZL) in the expression of endothelial and osteogenic genes on interacting endothelial and mesenchymal stem cells, an issue that was not previously addressed. Alendronate and ZL, 10⁻¹²–10⁻⁶ M, were evaluated in a direct co-culture system of human dermal microvascular endothelial cells (HDMEC) and human bone marrow mesenchymal stem cells (HMSC), over a period of 14 days. Experiments with the respective monocultures were run in parallel. Alendronate and ZL caused an initial dose-dependent stimulation in the cell proliferation in the monocultures and co-cultures, and did not interfere with their cellular organization. In HDMEC monocultures, the expression of the endothelial genes CD31, VE-cadherin and VEGFR2 was down-regulated by AL and ZL. In HMSC monocultures, the BPs inhibited VEGF expression, but up-regulated the expression of the osteogenic genes alkaline phosphatase (ALP), bone morphogenic protein-2 (BMP-2) and osteocalcin (OC) and, to a greater extent, osteoprotegerin (OPG), a negative regulator of the osteoclastic differentiation, and increased ALP activity. In co-cultured HDMEC/HMSC, AL and ZL decreased the expression of endothelial genes but elicited an earlier and sustained overexpression of ALP, BMP-2, OC and OPG, compared with the monocultured cells; they also induced ALP activity. This study showed for the first time that AL and ZL greatly induced the osteogenic gene expression on interacting endothelial and mesenchymal stem cells.     

Autonomous migration of human fetal skin fibroblasts into a denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen

Summary Human fetal skin fibroblasts (TIG-3S) were found to migrate into a denuded area in a cell monolayer when cultured in both serum-depleted and serum-supplemented media, unlike adult-donor skin fibroblasts which migrated well only when cultured in serum-supplemented medium. Therefore, a series of experiments was carried out to determine whether autocrine factors are involved in their migration. The migration of TIG-3S cells in serum-depleted medium was suppressed by the addition of suramin, a factor with growth factor antagonist properties, which suggests that growth factors are important for cell migration. The suramin-induced inhibition was reversed completely by adding excess basic fibroblast growth factor (bFGF) to the culture medium and partially by platelet-derived growth factor (PDGF). Treatment with neutralizing anti-PDGF antibody did not suppress TIG-3S cell migration, whereas neutralizing anti-bFGF antibody did, which indicates that bFGF is an autocrine and PDGF a paracrine factor involved in cell migration. Next, an experiment was performed to ascertain whether the extracellular matrix is involved in TIG-3S cell migration. Monensin, an inhibitor of extracellular matrix secretion, inhibited cell migration, which was reversed by adding excess type I collagen, but not excess plasma fibronectin. In addition, further evidence for the involvement of collagen was provided by the observation that ethyl-3,4-dihydroxybenzoate, a specific inhibitor of collagen synthesis, suppressed cell migration. These results suggest that the autonomous migration of TIG-3S human fetal skin fibroblasts is mediated by bFGF and type I collagen, which they produce and secrete.     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Development of isolation and cultivation method for outer root sheath cells from human hair follicle and construction of bioartificial skin

Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites. Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS cells is 2.1×10³ cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA (0.02%) solution for 15 min at 37°C, however, our modified method was able to obtain about 6.9×10³ cells/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution. Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0×10⁷ cells was obtained in a serum-free medium, while a modified E-medium with mitomycin C-treated feeder cells produced a total of 6.3×10⁷ cells over 17 days when starting with 7.5×10⁴ cells. Finally, we confirmed the effectiveness of our ORS cell isolation method by presenting their ability for reconstructing the bioartificial skin epitheliumin vitro