Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells

Summary Studies on the regulation of differentiation in airway epithelial cells have been hampered by the lack of cell culture systems that differentiate in vitro. One such system that does exhibit differentiation is hamster tracheal epithelial cells (HTE). A major problem with this system, however, is that at the time cells differentiate, they lyze the collagen gel upon which they grow, resulting in termination of the culture. Here we report that by growing the HTE cells at 32° instead of 37°C we can totally prevent lysis of the collagen gel. Cells grown at this lower temperature maintain their differentiated phenotype as evidenced by abundant mucus granules and the secretion of authentic mucus glycoproteins into the culture media. We have also developed a method for subculturing the primary cells which allows growth and differentiation in secondary culture. The HTE cells were capable of being passaged at least three times and did not become transformed as judged by their inability to grow in soft agar and to produce tumors in syngeneic animals. This improved HTE cell culture system will allow detailed studies on the mechanisms which regulate growth, differentiation, and mucus secretion in surface airway epithelial cells. This work was supported in part by grants HL-19717 and HL-36854 from the National Institutes of Health, Bethesda, MD.     

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summary l-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo. This study was supported by research grants AM27451 and NS06277 from the National Institutes of Health and by a Research Center Grant from the Muscular Dystrophy Association.     

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.     

Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence

The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.     

CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures

Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.     

Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

Immuno-isolation and culture of biliary epithelial cells from normal human liver

Summary Biliary epithelial cells (BEC) lining the intra-hepatic biliary ducts are the site of damage in several immunologically mediated liver diseases. BEC are difficult to isolate since they represent only 5% of the total cell number in normal liver. In this communication, a novel method for their isolation from normal liver is presented using a monoclonal antibody (HEA125) with specificity for an epithelial cell surface glyco-protein reported to be expressed in liver only by biliary epithelium. By combining differential density centrifugation and immuno-magnetic separation using HEA125 pure BEC (10⁵ cells/g fresh tissue) were prepared routinely. These cells were maintained in culture for up to 4 weeks with significant increases in cell numbers. The ability to prepare BEC from human liver offers an opportunity to develop In Vitro models to investigate the aetiology of diseases in intra-hepatic biliary epithelium. EDITOR’S STATEMENT This is a novel application to purification of specific liver cell types directly from tissue. It is well-suited for rapid communication because of its novelty and potential utility to investigators.     

Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6–8.2 than at pH 6.8–7.2. The pH-associated increase in collagenase was parallel by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.     

Isolation and primary cultures of human intrahepatic bile ductular epithelium

Summary A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments.     

Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×10⁶ cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×10⁶ or at 0.25×10⁶ cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V _max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution. The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD, and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD.     

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.     

Cultured human skin fibroblasts absorb65Zn

The radioactive isotope⁶⁵Zn was used to study the incorporation of zinc by cultured human skin fibroblasts. The development of the method for studying cell uptake of⁶⁵Zn in a minimal synthetic medium is presented. Kinetics carried out on control cultures up to 240 min indicated that zinc uptake occurred in three phases, the first being the most rapid. Temperature and pH affect zinc uptake, in favor of an active transport process. In addition, the rate of incorporation is considerably decreased during the first phases after adding potassium cyanide, during the last phases after adding sodium iodoacetate, and during all the phases if dithioerythritol is used. A hypothesis is therefore proposed according to which several types of mechanisms would be involved in zinc uptake by fibroblasts. At least a part of these mechanisms is energy-dependent.     

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts.The human skin fibroblast cell populations were metabolically pulse labeled with ¹⁴C-galactose (48 h). The amounts of ¹⁴C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by -scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns.The incorporation of ¹⁴C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.     

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.     

Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence

Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

Stress-induced responses of human skin fibroblasts in vitro reflect human longevity

Unlike various model organisms, cellular responses to stress have not been related to human longevity. We investigated cellular responses to stress in skin fibroblasts that were isolated from young and very old subjects, and from offspring of nonagenarian siblings and their partners, representatives of the general population. Fibroblasts were exposed to rotenone and hyperglycemia and assessed for senescence‐associated β‐galactosidase (SA‐β‐gal) activity by flow cytometry. Apoptosis/cell death was measured with the Annexin‐V/PI assay and cell‐cycle analysis (Sub‐G1 content) and growth potential was determined by the colony formation assay. Compared with fibroblasts from young subjects, baseline SA‐β‐gal activity was higher in fibroblasts from old subjects (P = 0.004) as were stress‐induced increases (rotenone: P < 0.001, hyperglycemia: P = 0.027). For measures of apoptosis/cell death, fibroblasts from old subjects showed higher baseline levels (Annexin V+/PI+ cells: P = 0.040, Sub‐G1: P = 0.014) and lower stress‐induced increases (Sub‐G1: P = 0.018) than fibroblasts from young subjects. Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from young subjects (P = 0.017 and 0.006, respectively). Baseline levels of SA‐β‐gal activity and apoptosis/cell death were not different between fibroblasts from offspring and partner. Stress‐induced increases were lower for SA‐β‐gal activity (rotenone: P = 0.064, hyperglycemia: P < 0.001) and higher for apoptosis/cell death (Annexin V+/PI? cells: P = 0.041, Annexin V+/PI+ cells: P = 0.008). Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from offspring (P = 0.001 and 0.024, respectively) whereas rotenone‐induced decreases were lower (P = 0.008 and 0.004, respectively). These data provide strong support for the hypothesis that in vitro cellular responses to stress reflect the propensity for human longevity.