Cultures Of Fibroblast-like Cells Research Articles

Changes in the mitochondrial function of fibroblast-like cells exposed to copper oxide nanoparticles

Introduction. Mitochondria are targets for almost all types of damaging agents, including toxins and oxidative stress. There is no doubt that possible effects on mitochondria should be taken into account in a comprehensive assessment of metal toxicity. Our objective was to establish changes in the mitochondrial function under the effect of copper oxide nanoparticles in vitro.
 Material and methods. A monolayer culture of human lung fibroblast-like cells of the FLECH-104 line were exposed to copper oxide nanoparticles (CuO NPs) 21±4 nm in size, final concentrations of which in the media were 25, 50, and 100 μg/mL. We measured the rate of oxygen consumption by the cells and its changes under the influence of modulators, such as oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and rotenone combined with antimycin A. We estimated parameters of mitochondrial function and the bioenergetics index.
 Results. At the concentration of 100 μg/mL in the incubation medium, CuO NPs induced changes in the culture of fibroblast-like cells that impeded further assessment of the mitochondrial function. At the lower concentrations of 25 and 50 μg/mL, we observed a dose-dependent trend toward a decrease in ATP-related respiration and bioenergetics index. It is noteworthy that the concentration of 25 μg/mL increased the maximum and reserve respiratory capacity of the cells, which was probably related to the dual effect of copper as a toxicant and an essential element.
 Limitations of the study. The study was conducted using only one cell line and three concentrations of CuO nanoparticles suspended in the culture medium.
 Conclusion. We established that copper oxide nanoparticles, when added to the incubation medium, have a multidirectional effect on the mitochondrial function of fibroblast-like cells potentially attributed to biotic properties of this metal.

About 4-day rhythm of proliferative activity of fibroblast-like cell cultures isn\u2019t endogenous and don\u2019t depend from the variations of Earth\u2019s magnetic field

A study of the 4-day rhythm of the proliferative activity of the embryonic fibroblast-like cells in the logarithmic growth phase was carried out. It was shown that in cell cultures obtained on different days from embryos of different ages, the phase of the 4-day rhythm coincides. In vitro the maxima of the proliferative activity were consistent with the minima of the motor activity of mice. Freezing the culture for 2 or 6 days does not cause a shift in the phase of the 4-day rhythm of cell proliferative activity compare with the unfreezing culture. That indicates the existence of an external synchronizer, which determines the 4-day infradian rhythm of the proliferative activity of embryonic cells. Then we daily thawed samples of single L929 culture of mice fibroblast-like cells for 22 and 17 days and researched the dynamics of its proliferative activity. We also showed 4-day rhythm of the simultaneous increase in the number of cells for all thawed samples. Taking into account that deep freezing of a culture leads to the cessation of all life processes, the fact we obtained indicates an exogenous mechanism of the formation of about a 4-day rhythm of the proliferative activity of cell culture. Variations of the Earth's magnetic field could be one of the external synchronizers of the infradian rhythm. We studied the increase in number of L929 cell in conditions of a magnetic permalloy screen and showed that the magnetic shielding no affect the parameters of the infradian rhythm of L929 cell proliferative activity. So further searches of the external synchronizers are need.

Polyvinyl alcohol and pectin blended films: Preparation, characterization, and mesenchymal stem cells attachment.

The design of novel wound dressings for chronic wound treatment is still of great importance. One of the promising approaches is application of mesenchymal stem cells (MSCs), immobilized on a flexible polymer film, for healing. In this study, blended films based on polyvinyl alcohol (PVA) and pectin with different component ratio have been prepared by solution casting method and evaluated. Physicochemical properties of the formed PVA/pectin films, including their morphology, wettability, swelling, stability, mechanical characteristics, have been studied. We demonstrated that the surface of PVA/pectin films could be modified by ultraviolet or dielectric barrier discharge plasma exposure. After both ultraviolet and plasma treatment, the hydrophilicity of PVA/pectin films increased. It has been shown that additional crosslinking of PVA/pectin films with glutaraldehyde resulted in reinforcement of their structure. MSCs were cultured on neat and modified PVA/pectin samples to evaluate the effects of film characteristics and composition on cell behavior. It has been determined that MSCs effectively adhered to glutaraldehyde-crosslinked PVA/pectin films and formed on them the monolayer culture of fibroblast-like cells. The additional modification of PVA/pectin films with collagen resulted in enhancement of MSCs adhesion. Our results show that the obtained PVA/pectin films with adhered MSCs can be suggested for potential application as a part of novel complex wound dressings.

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures

IntroductionPeriprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures.MethodsPPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR.ResultsFibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased.ConclusionThe multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures.

Infradian Rhythm in Proliferative Activity of a Culture of Embryonic Fibroblast-Like Cells from C57BL/6 Mice

We studied daily dynamics of proliferative activity of embryonic fibroblast-like cells in culture during the logarithmic growth phase. Daily increase in cell count in the culture showed a 4-day rhythm that persisted over 3 weeks of culturing. In cultures from different animals, the phase of this rhythm was synchronous and did not depend on the moment of cell isolation. It can be hypothesized that the 4-day biorhythm of proliferative activity of embryonic fibroblast-like cells in culture is determined by external environmental factors, probably of electromagnetic nature.

Layer‐by‐layer buildup of polysaccharide‐containing films: Physico‐chemical properties and mesenchymal stem cells adhesion

Layer-by-Layer assembled polyelectrolyte films offer the opportunity to control cell attachment and behavior on solid surfaces. In the present study, multilayer films based on negatively charged biopolymers (pectin, dextran sulfate, carboxymethylcellulose) and positively charged polysaccharide chitosan or synthetic polyelectrolyte polyethyleneimine has been prepared and evaluated. Physico-chemical properties of the formed multilayer films, including their growth, morphology, wettability, stability, and mechanical properties, have been studied. We demonstrated that chitosan-containing films are characterized by the linear growth, the defect-free surface, and predominantly viscoelastic properties. When chitosan is substituted for the polyethyleneimine in the multilayer system, the properties of the formed films are significantly altered: the rigidity and surface roughness increases, the film growth acquires the exponential character. The multilayer films were subsequently used for culturing mesenchymal stem cells. It has been determined that stem cells effectively adhered to chitosan-containing films and formed on them the monolayer culture of fibroblast-like cells with high viability. Our results show that cell attachment is a complex process which is not only governed by the surface functionality because one of the key parameter effects on cell adhesion is the stiffness of polyelectrolyte multilayer films. We therefore propose our Layer-by-Layer films for applications in tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2093-2104, 2018.

38 ROCKY MOUNTAIN BIGHORN SHEEP (OVIS CANADENSIS CANADENSIS) EMBRYOS PRODUCED USING SOMATIC CELL NUCLEAR TRANSFER

The present objective was to determine if interspecies somatic cell nuclear transfer (SCNT) could be a viable option for producing cloned Rocky Mountain bighorn sheep (RMBS) embryos, a model for other endangered ovine species or sheep of high genetic value. A primary question was whether viable cells from a harvested 5.5-year-old male RMBS (Ovis canadensis canadensis) could be cultured 4 days postmortem for SCNT embryo production. Domestic sheep ovaries from an abattoir were placed in TCM199 holding medium containing Hanks’ salts, kept at ambient temperature, and transported to the laboratory in less than 3 h. Oocytes were collected using 2 methods, aspirating visible follicles (G1) or slicing ovary tissues (G2), and matured in TCM199-based medium. At 20 h postmaturation, oocytes were stripped of cumulus cells and those with an extruded polar body underwent SCNT using ViaGen's protocol. Tissue samples were collected from the RMBS, placed in plastic bags, and shipped to the laboratory for cell culture; that is, tissue disinfection and mincing, placement in cell culture flasks with cell culture medium and into an incubator at 38.5°C in a humidified atmosphere of 5% CO2. At confluency, cells having fibroblast-like cell morphology were cryopreserved using DMSO-containing medium and stored at –196°C. For SCNT, 4-day postmortem donor cells were thawed and cultured for at least 3 days. Matured oocytes were enucleated using a micromanipulation system and Hoechst 33342 to ensure nucleus removal. A donor cell was inserted into an enucleated cytoplast and fusion pulses were applied to reconstruct embryos. Cloned embryos were cultured in modified SOF in 5% CO2, 5% O2, and 90% N2 for 6 days before shipment in a portable incubator at 38.5°C in SOF for analysis on Day 7. Pearson's χ2 test and Fisher's exact test were used to determine statistical differences (α = 0.05). The 60 ovaries yielded 324 oocytes (5.4 oocytes/ovary) and 217 matured (67%) of which 204 were enucleated, 156 fused (77%), and 102 cleaved (65%), producing 25 blastocysts and morulae (25/156 = 16%). G1 resulted in 62 cleaved embryos of 91 fused, whereas G2 resulted in 40 cleaved embryos of 65 fused (χ2 = 0.728; P = 0.393). Using the IETS stage and grading system, 3 blastocysts (one 5–2, one 6–1, one 6–2) and 22 morulae (4–1) appeared viable, but the rest had degenerated. Specifically, G1 produced 3 blastocysts and 8 stage 4–1 embryos versus 14 stage 4–1 embryos in G2 (based on fused or cleaved embryos: χ2 = 2.516, P = 0.113 and χ2 = 3.914, P = 0.048, respectively). Fibroblast-like cell cultures were obtained from 4- and 5-day postmortem tissue samples; however, only Day 4 cultured cells were used for RMBS SCNT embryo production. Although G1 produced more blastocysts than G2, the difference was not statistically significant (P > 0.05).

Антипроліферативна активність ранібізумабу, бевацізумабу та пегаптанібу в культурі фібробластоподібних клітин in vitro

Антипроліферативна активність ранібізумабу, бевацізумабу та пегаптанібу в культурі фібробластоподібних клітин in vitro

In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4°C for different time intervals

Live animals have been produced recently from animal tissues preserved for decades at frozen temperatures with or without cryoprotectants. However, the tissues in these studies were cryopreserved within few hours of animal death to obtain culturable live cells as nuclear donors. How long the tissues can be left unfrozen after animal death, without losing the viability and potential to in vitro culture with comparable morphology and proliferative rate as the fresh tissues, is not completely understood. To understand this phenomenon, ear skin samples from individual sheep (n=3) were procured from slaughter plant and stored at 4 °C. After various intervals (2, 8, 24, 32, 48, and 56 h after slaughter), 2-3 mm(2) pieces (n=10) of skin samples were cultured for 12 d on two dishes (60 mm) for each sheep. Outgrowth of fibroblast-like cells was observed as early as day 4 of culture and was visible on dishes of all time points including 56 h by day 10. The number of outgrowing cells decreased with increasing time interval between animal slaughter and culture initiation. Secondary cultures were successfully established for all the time points. All cultures proliferated well and were apparently normal. Passage 2 cultures of 2 h and 56 h interval for one of the three sheep were compared for their growth pattern and proliferation rates. The population doubling time of 2 h and 56 h intervals was 33.12 and 34.8 h, respectively, and both the lines exhibited similar cell morphology and an "S"-shaped growth curve. These results suggest that skin tissues of sheep and perhaps other animal species with superior traits are effectively preserved at cellular level at least for 56 h at normal refrigerating conditions, without need of complicated cryopreservatives/cryotanks that are usually not available at small farms.

Pulsed electromagnetic fields decrease proinflammatory cytokine secretion (IL-1β and TNF-α) on human fibroblast-like cell culture

The clinical use of pulsed electromagnetic fields (PEMF) in osteoarticular pathology is widely extended, although the mechanisms involved are unknown. The aim of this study was to evaluate the action of a new protocol of treatment with PEMF on liquid medium cultures of fibroblast-like cells derivates of mononuclear peripheral blood cells. Fibroblast-like cells growth was obtained in liquid medium culture from mononuclear cells (MNC) of human peripheral blood. The PEMF irradiation protocol included an intensity of 2.25mT, a frequency of 50Hz and an application time of 15min on days 7, 8 and 9 of cell culture. Immunophenotype was performed with specific heterologous monoclonal antibodies for each cell receptor (Vimentin, Cytokeratin, CD34, CD41, CD61 and CD68). The cytokines' production was determined in the supernatant of the culture medium by means of the Luminex technology. The immunophenotype did not show any statistical difference on comparing treated against non-treated cell cultures on any of the days. In the treatment cell population, the proinflammatory cytokines, IL-1β and TNF-α showed a significant decrease on days 14 and 21 of the culture, whilst IL-10 increased significantly on day 21. It is concluded that PEMF irradiation does not alter the cell immunophenotype of the fibroblast-like cell population, but does provoke a decrease in the production of inflammatory-type cytokines (IL-1β, TNF-α) and an increase in cytokines of lymphocytic origin (IL-10). These facts coincide with the chronology of the clinical effect undergone by patients with osteoarticular pathology after PEMF irradiation.

Cytogenetic Characterization and Fluorescence in situ Hybridization of (GATA)10 Repeats on Established Primary Cell Cultures from Indian Water Snake (Natrix piscator) and Indian Mugger (Crocodylus palustris) Embryos

Sex determination among reptiles has continued to draw the attention of geneticists and the mechanisms involved have been extensively studied and documented in the past 3 decades. The setting up of primary cell lines of reptilian tissues is an important tool in the present study which is a unique aspect not applied in earlier studies. Establishing the cell lines from various species of reptiles would help in our understanding of the mechanisms of evolution and differentiation of sex chromosomes. Therefore, in the present study, we have established for the first time primary cell cultures from Indian water snake (Natrix piscator) and Indian mugger (Crocodylus palustris) embryos. In the preliminary growth stage, 2 types of cells, fibroblast- and epithelial-like, were found to be attached and proliferating in vitro. These fibroblast-like cell cultures were later overtaken by epithelial cells. The cell lines were grown in minimal essential medium supplemented with fetal bovine serum and subcultured for a period of 8–10 months. The morphology of cell types was kept under constant observation microscopically. Interestingly, at a subsequent passage of the cells sporadically scattered neuronal-like and beating cells were observed. The suitable temperature for growth of these cell cultures was 28–30°C. Chromosome analysis was performed from the actively proliferating cells, which revealed 5 pairs of macrochromosomes and 15 pairs of microchromosomes in Natrix piscator, and 15 pairs of only macrochromosomes in Crocodylus palustris. (GATA)_n repeats are well known to be associated with sex chromosomes. Fluorescence in situ hybridization performed with (GATA)₁₀ repeats delineated the W chromosome in the cells of Natrix piscator which has so far not been reported. This cell culture method has presently only been applied to water snakes and crocodile embryos in the current study, but it will be employed in other reptilian species and could go a long way to being a sustainable source of primary cells. This would eventually serve as an important tool for molecular studies in reptiles and other species in the future.

3,3′-Diindolylmethane attenuates experimental arthritis and osteoclastogenesis

3,3′-Diindolylmethane (DIM) is a natural compound formed during the autolysis of glucobrassicin present in Brassica food plants. This study aimed to investigate the therapeutic efficacies of DIM on experimental arthritis. The effects of DIM on experimental arthritis were examined on a rat model of adjuvant-induced arthritis (AIA), with daily AIA paw swelling observation and histological/radiographic analysis. To elucidate the possible mechanisms of its action, serum cytokine levels as well as the expression of receptor activator for nuclear factor κ B ligand (RANKL) in infected tissues were subsequently analyzed. The impact of DIM on osteoclastogenesis was further investigated on a mouse model of endotoxin-induced bone resorption (EIBR) and in vitro cultures of fibroblast-like cells and osteoblasts, with RANKL expression being evaluated with great interest. The administration of DIM was demonstrated to attenuate AIA in animal models, as judged by clinical and histologic indices of inflammation and tissue damage. On the one hand, DIM could reduce the expression of several inflammatory cytokines, which was, however, not adequate to prevent the development of the arthritis. On the other hand, DIM was shown to effectively inhibit the expression of RANKL, leading to the blockade of osteoclastogenesis and consequently an alleviation of experimental arthritis. Further in vitro and in vivo studies confirmed the inhibition of RANKL by DIM. DIM has shown anti-arthritis activity in animal models via inhibiting the expression of RANKL, and thus may offer potential treatments for arthritis and associated disorders.

Mesenchymal Stem Cells from Different Organs are Characterized by Distinct TopographicHoxCodes

Mesenchymal stem cells (MSC) are multipotent cells found as part of the stromal compartment of the bone marrow and in many other organs. They can be identified in vitro as CFU-F (colony forming unit-fibroblast) based on their ability to form adherent colonies of fibroblast-like cells in culture. MSC expanded in vitro retain characteristics appropriate to their tissue of origin. This is reflected in their propensity for differentiating towards specific lineages, and their capacity to generate, upon retransplantation in vivo, a stroma supporting typical lineages of hematopoietic cells. Hox genes encode master regulators of regional specification and organ development in the embryo and are widely expressed in the adult. We investigated whether they could be involved in determining tissue-specific properties of MSC. Hox gene expression profiles of individual CFU-F colonies derived from various organs and anatomical locations were generated, and the relatedness between these profiles was determined using hierarchical cluster analysis. This revealed that CFU-F have characteristic Hox expression signatures that are heterogeneous but highly specific for their anatomical origin. The topographic specificity of these Hox codes is maintained during differentiation, suggesting that they are an intrinsic property of MSC. Analysis of Hox codes of CFU-F from vertebral bone marrow suggests that MSC originate over a large part of the anterioposterior axis, but may not originate from prevertebral mesenchyme. These data are consistent with a role for Hox proteins in specifying cellular identity of MSC.

The innate immune system as a therapeutic target in arthritis

Rheumatoid arthritis is a chronic inflammatory disease that may be characterized by several phases. In the initial induction phase of the disease there may be a breech in the normal mechanisms that protect the host from destruction. In the later phase, pathways that are independent of the adaptive immune system may perpetuate inflammation. These pathways may be active in patients who are refractory to biologic therapies that target key cytokines, IL-1 and tumor necrosis factor alpha. The Toll-like receptors (TLRs) are an integral part of the innate immune system, and are present on synovial lining cells, as well as the cells of the immune system, which may amplify their contribution to the inflammatory cascade. We have found that TLR-4 and its coreceptor CD14 can modulate arthritis and can modulate inflammatory joint disease in murine models of arthritis. In particular, exposure to a TLR-4 ligand stimulates release of IL-6 from synovial fibroblast-like cells in culture, and circumvents the requirement for IL-1 receptor signaling in a serum transfer model of arthritis. These data suggest that triggering the innate immune response through the TLRs with either microbial or autologous ligands plays a role in the initiation or perpetuation of rheumatoid arthritis.

Chemically modified ribozyme targeting TNF-alpha mRNA regulates TNF-alpha and IL-6 synthesis in synovial fibroblasts of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is chronic polyarthritis in which a variety of inflammatory cytokines play a role. Since tumor necrosis factor-alpha (TNF-alpha) is one of the most important cytokines in the pathogenesis of RA, we evaluated the feasibility of ribozymes as a therapeutic agent to control the inflammatory process of RA synovium. A hammerhead ribozyme against TNF-alpha was chemically modified to increase nuclease resistance and added to RA fibroblastlike cell cultures without using a delivery system. The cellular uptake of fluorescent-labeled ribozyme into synovial cells was found to last at least 48 hr by confocal laser scanning microscopy. The ribozyme targeting TNF-alpha gene inhibited both the expression of TNF-alpha mRNA and the secretion of TNF-alpha and IL-6. The cytotoxic effect by the ribozyme on synovial cells was negligible when determined by an alamar blue assay. Chemically modified ribozymes designed to suppress the TNF-alpha gene may be potential as a therapeutic agent for rheumatoid arthritis.

THIAZOLIDINEDIONE INDUCES THE ADIPOSE DIFFERENTIATION OF FIBROBLAST-LIKE CELLS RESIDENT WITHIN BOVINE SKELETAL MUSCLE

To investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) in adipocyte formation within the skeletal muscle of beef cattle, fibroblast-like cells were isolated from the longissimus muscle of cattle and cultured with activators of murine PPARγA thiazolidinedione T-174, which is a specific ligand for PPARγ, stimulated adipose differentiation (evaluated by counting differentiated adipocytes under microscopic observation) in a dose-dependent fashion. A peroxisome proliferator Wy14,643 which strongly activates the α isoform of murine PPAR also stimulated differentiation but its potency was weaker than that of T-174. Unexpectedly, 15-deoxy-Δ12,14-prostaglandin J2, which is believed to be an endogenous ligand for PPARγ, could not induce adipose differentiation in doses which have been found to be effective on rodent cells. Immunoblotting analysis confirmed the significant expression of PPARγ protein in fibroblast-like cell cultures prepared from bovine skeletal muscle. In conclusion, bovine skeletal muscle contains adipose precursor cells expressing functionally active PPARγ.

CD34-Negative Hematopoietic Stem Cells Isolated from Human Peripheral Blood Cells as Ultimate Precursors of Hematopoietic Progenitors

<i>Background</i>: The earliest hematopoietic stem cell is still not yet identified. From experiments in dogs there is evidence that the earliest hematopoietic stem cell is a CD 34– fibroblast-like cell which can be isolated from bone marrow stromal cultures. In this set of experiments we wanted to identify such an early CD34– fibroblast4ike cell population from human peripheral blood. <i>Material and Methods</i>: Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteer donors and incubated with interleukin-6(IL-6)-containing medium in tissue culture flasks. After establishing an almost confluent adherent layer of fibroblast4ike cells which attach to the plastic surface, the cells were stimulated with various growth factors. The emerging nonadherent cells as well as the adherent cells were applied to a colony-forming unit (CFU) assay in semisolid agar and to long-term culture-initiating cell (LTC-IC) assay. <i>Results:</i> CD34– adherently growing cells could be isolated from human peripheral blood by IL-6-mediated plastic adherence. These cells grow as fibroblast-like cells in culture and differentiation can be induced by stem cell factor (SCF), while IL-6 induces proliferation of these cells. While differentiating, CD34– fibroblast-like cells lose the ability to grow adherently and start to express CD34 and eventually HLA-DR. The differentiating CD34+ cells are also capable of producing CFU and LTC-IC. <i>Conclusion:</i> This set of experiments provides evidence that the earliest identifiable hematopoietic stem cell is a CD34– adherently growing fibroblast-like cell which is part of the marrow microenvironment in vivo and can differentiate into hematopoietic progenitors upon appropriate growth factor signaling. This kind of early stem cell can also be isolated from PBMNC.

Vitronectin in Colorectal Adenocarcinoma—Synthesis by Stromal Cells in Culture

We have investigated the expression and cellular source of vitronectin in colorectal adenocarcinoma. Immunofluorescence staining of tissue sections revealed the presence of vitronectin in the stroma of the 11 tumors studied, but not in adjacent normal colon. A method was devised for the isolation from colorectal adenocarcinomas of fibroblast-like cells that stained positive for vimentin but negative for cytokeratin. These tumor-derived stromal cells synthesized and secreted vitronectin, as revealed by metabolic labeling and immunoprecipitation. This was confirmed by Southern blot analysis of polymerase chain reaction amplification products from reverse-transcribed RNA. Normal skin fibroblasts did not synthesize vitronectin. Immunofluorescence staining showed vitronectin deposited at focal contact sites in the tumor-derived cells, where it colocalized with vinculin and the αv integrin subunit. The deposition of vitronectin into focal contact sites was not dependent on the presence of serum. The finding that vitronectin can be synthesized and secreted by tumor-derived fibroblast-like cells in culture indicates that vitronectin expression can be promoted by as yet unknown signals provided in disease states, such as cancer.

Alterations in contact and density-dependent arrest state in senescent WI-38 cells

Normal human WI-38 fibroblast-like cells in culture undergo a process of senescence, one feature of which is a gradual decline in proliferative capacity. As these cells reach the end of their replicative life span they exhibit decreases in the fraction of cells able to synthesize DNA, in the number of doublings per passage (constant seeding density), and in the cell harvest and saturation densities. They also display increased average cell cycle times, largely at the expense of longer G1 intervals. These alterations are accompanied by morphologic changes, including cell enlargement. Before the end of the replicative life span or phase-out, there is a highly reproducible (55/58 sublines) cell loss of approximately 50%; however, a stable population survives that can exist in a viable yet nonproliferative state for many months. This stable population maintains an extremely low saturation density, representing < 5% of that achieved by early passage cultures. Further, we show that maximum harvest densities achieved by senescent cells are lower, irrespective of seeding densities, i.e. when placed at cell densities higher than those normally achieved by senescent cultures they display a net decline in cell number. This decline continues until the cell density approximates the density that would have been achieved had the cultures been seeded at standard density (1 x 10(4) cells/cm2).(ABSTRACT TRUNCATED AT 250 WORDS)

Replicative senescence of human fibroblast-like cells in culture.

The life history of fibroblast and fibroblast-like cells includes an initial stage of outgrowth and establishment in culture; a period of vigorous proliferation which has a variable length, depending on the tissue of origin, age of the donor, etc.; a period of declining proliferative vigor which includes substantial cell death; and finally, the emergence of an (apparently) long-lived population which is unable to proliferate in response to growth factors. During the phase of declining proliferative vigor, the cells acquire characteristics, some of which are similar to the characteristics of cells in older individuals. Eventually the culture completely loses proliferative capacity. A comparable life history has been described for glial cells, keratinocytes, vascular smooth muscle cells, endothelial cells, and lymphocytes which suggests that this life history is characteristic of those cell types that, in vivo, retain the capacity for proliferation throughout the life span. Numerous studies have shown a correlation between the age of the tissue donor and the replicative life span of the cells in culture. In addition, for a small sample of species, there is a direct correlation between fibroblast replicative life span in vitro and maximum life span potential of the species. The period in the life history that is usually referred to as the "senescent phase" is probably more complicated than was originally thought, since studies with life span modulators suggest that there is a "conditionally" senescent state from which cells can be rescued for one or more additional rounds of DNA synthesis. Finally, the cells enter an "obligatory" arrested state in which only SV40 infection can reverse the block to DNA synthesis but not the block to mitosis. The modern era of aging research in tissue culture is just over 30 years old. The inception of the field really began with the recognition by Hayflick and Moorhead (109) that the phenomenon of senescence in vitro paralleled, in some of its characteristics, cell aging in vivo and thus provided a model that could be used to study the cellular mechanisms underlying senescence in controlled environmental conditions. The research in this area began with a detailed characterization and comparison of young versus senescent cell morphology and physiology. These studies provided the basis for a wide variety of subsequent studies that addressed possible mechanisms underlying cell senescence. These included studies on DNA repair, protein synthetic errors, chromatin structure and function, and mechanisms for modulating replicative life span.(ABSTRACT TRUNCATED AT 400 WORDS)