Subcultured Fibroblasts Research Articles

Bio-artificial pleura using autologous dermal fibroblast sheets to mitigate air leaks during thoracoscopic lung resection

Lung air leaks (LALs) due to visceral pleura injury during surgery are a difficult-to-avoid complication in thoracic surgery (TS). Reliable LAL closure is an important patient management issue after TS. We demonstrated both safeties of transplantation of a cultured human autologous dermal fibroblast sheet (DFS) to LALs. From May 2016 to March 2018, five patients who underwent thoracoscopic lung resection met all the inclusion criteria. Skin biopsies were acquired from each patient to source autologous dermal cells for DFS fabrication. During the primary culture, fibroblasts migrated from the dermal tissue pieces and proliferated to form cell monolayers. These fibroblasts were subcultured to confluence. Transplantable DFSs were fabricated from these subcultured fibroblasts that were trypsinized and seeded onto temperature-responsive culture dishes. After 10 days of fabrication culture, intact patient-specific DFS were harvested. DFSs were analyzed for fibroblast cell content and tissue contaminants prior to application. For closing intraoperative LAL, mean number of transplanted autologous DFS per patient was 6 ± 2 sheets. Mean chest drainage duration was 5.0 ± 4.8 days. The two patients with major LAL had a drainage duration of more than 7 days. All patients currently have no LAL recurrence after discharge. DFSs effectively maintain LAL closure via remodeling of the deposited extracellular matrix. The use of autologous DFSs to permanently close air leaks using a patient-derived source is expected to reduce surgical complications during high-risk lung resections.

Preclinical Safety Studies on Autologous Cultured Human Skin Fibroblast Transplantation

Recently, FDA approved the clinical use of autologous fibroblasts (LAVIV™) for the improvement of nasolabial fold wrinkles in adults. The use of autologous fibroblasts for the augmentation of dermal and subcutaneous defects represents a potentially exciting natural alternative to the use of other filler materials for its long-term corrective ability and absence of allergic adverse effects proved by clinical application. However, compared to the clinical evidence, preclinical studies are far from enough. In this study, human skin-derived fibroblasts were cultured and expanded for both in vitro and in vivo observations. In vitro, the subcultured fibroblasts were divided into two groups. One set of cells underwent cell cycle and karyotype analysis at passages 5 and 10. The second group of cells was cocultured in medium with different concentrations of human skin extract D for the measurement of collagen concentration and cell count. In vivo, the subcultured fibroblasts were injected into nude mice subcutaneously. Biopsies were taken for morphology observation and specific collagen staining at 1, 2, and 3 months after injection. The results in vitro showed no significant differences in cell cycle distribution between passages 5 and 10. Cell proliferation and secretion were inhibited as the concentration of extract D increased. In vivo, the fibroblasts were remarkably denser on the experimental side with no dysplastic cells. Mitotic cells were easily observed at the end of the first month but were rare at the end of the third month. Type III collagen was detected at the end of the first month, while collagen type I was positive at the end of the second month. The content of both collagens increased as time passed. The above results indicated that the use of the autologous fibroblasts was safe, providing a basic support for clinical use of fibroblasts.

Multiplex analyses of cytokine and chemokine release from the cultured fibroblast of nasal polyps: the effect of IL-17A

Conclusion: Our results demonstrate for the first time a potentially enhanced basal secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-17A-stimulated secretion of IL-6 from nasal polyp fibroblasts, enhanced basal secretion of IL-6 from eosinophilic nasal polyp fibroblasts, and a remarkable up-regulation of IL-9 and granulocyte colony-stimulating factor (G-CSF) from nasal fibroblasts by IL-17A stimulation. Objectives: The fibroblast, one of the main cell types making up nasal polyps, is thought to be a target cell of various cytokines. Methods: Subcultured fibroblasts were established from human polyp biopsy tissues. Simultaneous quantification of 27 kinds of cytokines and chemokines in culture supernatants in unstimulated and IL-17A-stimulated conditions was performed with a human multiplex cytokine assay system. Results: The IL-17A receptor was expressed at similar levels in all three groups. In the eosinophilic group, basal secretion levels of IL-6 were significantly higher than those in the control and non-eosinophilic groups. Basal secretion of MCP-1 in both the non-eosinophilic and eosinophilic groups was also higher than that of the control group. Both IL-9 and G-CSF secretion were remarkably enhanced by IL-17A stimulation in all three groups. The receptor-mediated response by IL-17A significantly up-regulated IL-6 release alone in the non-eosinophilic and eosinophilic groups as compared with the control group.

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I-III, II-III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 micromol 1(-1)) were added. Results indicated that ascorbate at 100 micromol 1(-1) was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 micromol 1(-1) however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I-III and II-III activities which required 300 micromol 1(-1) ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations.

Increased sensitivity to bleomycin in upper aerodigestive tract mucosa of head and neck squamous cell carcinoma patients

Previous studies on lymphocytes have suggested that patients with head and neck squamous cell carcinoma (HNSCC) have an increased susceptibility for chromosomal damage induced by bleomycin, a known radiomimetic mutagen. However, it has so far not been possible to study whether this genetic instability is present also in the epithelial component of the upper aerodigestive tract mucosa, the tissue from which HNSCC originates. In the present study, we have successfully cultured epithelial cells and fibroblasts isolated from non-neoplastic mucosa samples of 30 HNSCC patients and 56 controls. All cell cultures were exposed to bleomycin and chromosome instability was assessed by analysis of chromosome breakage in cells harvested after 2 h of exposure and subsequent removal of bleomycin. Furthermore, the status of the fragile histidine triad gene (FHIT) in chromosome band 3p14.2 was studied by fluorescence in situ hybridization (FISH) in epithelial cells that had been cultured after removal of bleomycin. Chromosomal damage, in the form of chromosomal breaks and gaps, was seen in all cell cultures harvested 2 h after exposure to bleomycin. In epithelial cells, the frequency of chromosome breakage was significantly higher among HNSCC patients than among controls [mean breaks per cell (b/c) 1.02 vs. 0.77, p = 0.02]. When subdivided according to smoking status, age, and sex, a significantly higher frequency of chromosome breakage was still found in HNSCC patients (smokers, p = 0.01, age ≤ 70 group, p = 0.03, male, p = 0.02). However, no significant difference was found between fibroblasts from HNSCC patients and controls (b/c 1.21 vs. 1.23). In the cell cultures growing after termination of bleomycin exposure, the frequency of chromosome breakage was generally very low and no significant difference could be found between the HNSCC patients and controls when epithelial cells were examined. In subcultured fibroblasts, a higher frequency was found in HNSCC patients than in controls (b/c 0.59 vs. 0.19, p = 0.03). Interphase FISH on cultured epithelial cells from HNSCC patients ( n = 10) and controls ( n = 12) showed that the frequency of FHIT deletion was significantly higher in HNSCC patients than in controls. Our results support the notion that HNSCC patients accumulate genetic damage more rapidly, possibly due to an inherent susceptibility, which could explain the high risk for multi-focal neoplastic cell transformation.

Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor-α on Corneal Myofibroblast Apoptosis

Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-alpha in corneal myofibroblast apoptosis was explored. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm2). Mouse anti-alpha-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha receptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF-alpha (20 ng/mL), and TNF-alpha+cPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope. Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF-alpha receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF-alpha for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, demonstrating a synergistic action between PAF and TNF-alpha. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF. Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF-alpha suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.

Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop.

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.

Enzymatic Variations Related to Glucose and Glycogen Catabolism in Serially Subcultured Human Fibroblasts

The specific activities of some glucose and glycogen degradative enzymes were studied in serially subcultured fibroblasts harvested at the same state of growth and replication. A continuous increase i

Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts.

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2'-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.

Adhesion of hematopoietic cells and lymphocytes to stromal mechanocytes in vitro

Adhesion of guinea pig bone marrow and spleen cells to stromal mechanocytes of medullary, splenic, and thymic origin, to peritoneal exudate fibroblasts, and also to macrophages was studied in monolayer cultures. Primary cultures and subcultures of fibroblasts were able to bind hematopoietic cells and lymphocytes, and this property was independent of the origin of the mechanocytes. Hematopoietic cells were much less adherent to macrophages. Adhesion of cells to mechanocytes is determined by the number of adhesion sites present on the surface of the fibroblasts. There are significantly more of these sites for adhesion of myeloid cells on the surface of stromal mechanocytes than for adhesion of lymphocytes.

Aging in vitro: Growth of cultured cells from the Galapagos tortoise

Skin biopsies from two growing and two fully-grown Galapagos tortoises, a species that lives twice as long as man, were explanted in vitro. Cellular outgrowth was more vigorous from explants of young tortoises but epithelial cells stopped dividing early in the history of all cultures, even at the optimum temperature of 30 °C. Serially subcultured fibroblasts from young tortoises divided more rapidly and achieved longer lifespans than fibroblasts from old tortoises in terms of mean population doublings and to a lesser extent, calendar time. For all cultures the lifespans exceeded those reported for human diploid fibroblasts. The results indicate that a proportionality exists between the potential (remaining) lifespan in vivo and the mitotic capacity of cultured diploid cells in vitro.

In Vitro-effects of vitamin A acid

The in vitro-effect of vitamin A acid (VAA) upon DNA synthesis was studied in three different cell types: lymphocytes, epidermal cells grown as primary cultures and subcultured fibroblasts. The incorporation of3H-TdR after exposure of the culture to various concentrations of VAA was determined by liquid scintillation counting.

Production and release of acetylcholinesterase by cultured chick embryo muscle

The acetylcholinesterase (AChE) activity of cultures from 11-day-old chick embryo muscle cells was studied for up to 4 weeks in vitro. AChE activity was found in mononucleated cells and multinucleated myotubes. The activity increased greatly after fusion. Maximum AChE levels were reached after 7–10 days of incubation and tended to decline thereafter. Multiple forms of AChE found in embryo muscle in situ were present in cultures before and after fusion. Selective inhibitors and substrates were used to show that AChE was released by the cells into their medium. Within a 2-day period the AChE that accumulated in the medium averaged over 6 times that remaining in the cells. Release of AChE from the cells was inhibited by cycloheximide, and AChE levels in cells and medium were much reduced when differentiation was inhibited by bromodeoxyuridine. Little AChE was present in subcultures of fibroblasts from muscle cultures. Acetyl-β-methylcholine and, to a lesser degree, choline itself, prevented the decrease in AChE levels of 2- to 3-week-old muscle cultures.

Testosterone metabolism by serially subcultured fibroblasts from genital and nongenital skin of individual human donors

Summary Skin fibroblasts were cultured from the prepuce and deltoid area of three males, and from the labia majora and abdomen of two females. The genital skin strains metabolised testosterone much faster than those derived from nongenital skin. This is the first clear demonstration of the persistence in serially subcultured cells of a metabolic difference between nonmalignant somatic tissues of human beings.

Testicular feminization: Expression in sex skin fibroblast culture

The inheritance and pathogenesis of human testicular feminization (TF) are unknown. The possibilities of X-linked recessive inheritance and primary target organ refractoriness to testosterone (T) could be studied optimally in serially subcultured cell strains. Fibroblasts derived from sex and non-sex skin of males, females and patients with TF were incubated with T-4-14C. T and its metabolites in the culture medium were separated by paper chromatography and identified by reverse isotopic dilution. The rate of T metabolism was much greater in sex than in non-sex strains. For both newborn foreskin and adult labial strains, androstenedione was the primary metabolite to T, however they differed in their rate of production of 5α-androstanedione and andosterone. In a strain derived from the foreskin of a 7-year old, 5α-androstanedione, rather than andrastenedione, appeared to be the major early metabolite. 5α-dihydro-testosterone (DHT) was a major metabolite in none of the strains. Sex and non-sex strains from two patients with TF were indistinguishable: Both had low rates of T metabolism. We conclude that: 1) in situ differences in T metabolism between normal sex and non-sex skin persist in their serially subcultured fibroblasts; 2) an expression of the TF gene is detectable in sex skin fibroblasts without reference to the rate of DHT formation; and 3) Lyonization of cloned heterozygous fibroblasts may prove that human TF is X-linked.

Increase in survival of subcultured fibroblasts mediated by serotonin.

WE have already reported a cytospecific effect of serotonin in enhancing the growth of fibroblasts in vitro1, apparently by shortening the lag phase. We have now investigated this effect and the kinetics of cell growth in order to provide information about the mechanism of action of serotonin.