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At least 253 records · Page 14Linked to original sources
PubMed2004

Serial passage of MC3T3-E1 cells down-regulates proliferation during osteogenesis in vitro.

Generally, fibroblast-like cells and other types of human cells have been used to demonstrate the principles of replicative senescence in vitro and in vivo. These cells go through three stages of proliferation, including vigorous proliferation, declining proliferation and quiescence or no proliferation. Any variation of this process occurring in osteoprogenitor cells may offer insight into the mechanism of age-related osteopaenia that predisposes individuals to osteoporosis and bone fractures. We selected MC3T3-E1 cells derived from mouse calvaria to study the mechanism of replicative senescence of pre-osteogenic cells because: (i) these cells constitute a well-known model for studying osteogenesis in vitro; (ii) they undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture; and (iii) they show signs of replicative senescence. These cells were aged by multiple passaging before their use for studying growth kinetics and the effects of population density, effect of extracellular matrix (ECM), size and phases of the cell cycle. Our results show that (i) MC3T3-E1 cells go through the first two stages of proliferation in a manner similar to human cells, but escape the quiescent phase; (ii) the rate of proliferation is similar for low passage (LP) and high passage (HP) cells, but is decreased in very high passage cells (VHP); (iii) growth inhibition is observed using HP cells seeded at high density; (iv) HP ECM stimulates proliferation of both LP and HP cells; (v) a small increase in cell size is observed in HP cells, but no change is seen in the distribution analysis of their cell cycle; (vi) distribution analysis of the cell cycle of VHP cells reveals a decreased and an increased frequency of cells in S and G2 + M phases of their cell cycle, respectively. These results suggest that the mouse MC3T3-E1 cell line exhibits many of the cellular and molecular markers associated with replicative senescence in culture as defined by human cells, such as fibroblast-like cells. Alteration in the sensitivity of MC3T3-E1 cells to intercellular contact and increase in cell size are the primary factors contributing to decreased proliferation of HP cells.

Animals↗
PubMed1993

Pathological mineralization in a serially passaged cell line from rat pulp.

The ultrastructure of crystal formation in association with dental pulp cells isolated from rat incisor was studied in vitro. A clone, RPC-K, was obtained and incubated with Na-beta-glycerophosphate (BGP). Growing pulp cells showed low alkaline phosphatase (ALP) activity, which began to increase with cell proliferation. Pulp cells formed cell multilayers after day 14 of culture. Mineralized tissues were observed within cell multilayers on day 28 of culture. Vesicular structures were found around degenerate and necrotic cells. Some of these vesicles contained needle-like crystals. Organic structures appeared at the periphery of mineralized tissues with a post-embedding demineralization and staining method. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite (HAP). These findings suggest that the RPC-K cell line might be useful for a model system to investigate pathological mineralization.

Alkaline Phosphatase↗
PubMed1991

Antigenic stability of foot-and-mouth disease virus variants on serial passage in cell culture.

Two neutralizing monoclonal antibody (MAb)-resistant variants selected from an isolate of foot-and-mouth disease virus (FMDV) type A5 were repeatedly passaged in cell culture and monitored for susceptibility to neutralization by the selecting MAb. A variant isolated with a MAb to a conformational epitope (1-OG2) lost resistance in 20 passages, while a variant isolated with a MAb to a linear epitope (1-HA6) persisted for 30 passages. In both cases, the virus population emerging after passage was antigenically and genetically indistinguishable from the original wild-type parental virus (FMDV A5 Spain-86). Coinfection assays with the wild type and each variant, and between the variants, showed rapid conversion to a homogeneous population. Wild-type virus prevailed over the variants and for coinfection between the variants, the linear epitope variant 1-HA6. While both variants arose from a single nucleotide substitution and reversion to wild type occurred for each, it appears that the variant based on the continuous epitope (1-HA6) was more stable. We discuss the implications of these results for the antigenic diversity of FMDV and its relationship to virus evolution.

Animals↗
PubMed1968

Investigetion into the attenuation of influenza viruses by serial passage.

For vaccination live viruses are better than dead ones, but live influenza vaccines are difficult to prepare. One influenza A(2) and two influenza B viruses were passed in series in embryonated eggs. At several stages of their passage they were inoculated into volunteers, and their effects assessed by virus isolations, antibody rises, and clinical reactions. The A(2) virus and one of the influenza B viruses, both of which had grown readily in embryonated eggs on first isolation, continued to induce human infections and clinical reactions after 30 egg passes. The other influenza B virus acquired enhanced human pathogenicity after three passages from man to man. After adaptation to eggs in which it at first grew reluctantly, its human virulence was appreciably reduced. It underwent no further change during a total of 20 egg passes. There was little convincing evidence of an increased incidence of clinical reactions during the winter seasons, but the numbers of volunteers were too small to draw definite conclusions.

Animals↗
PubMed1996

Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.

The goal of our studies was to establish procedures for subculturing normal human tracheobronchial epithelial (NHTBE) cells without compromising their ability to differentiate into mucous and ciliated cells (i.e., differentiation competence) and to study the regulation of airway secretions by epidermal growth factor (EGF) and retinoic acid (RA). Primary NHTBE cells were obtained from a commercial source and subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for differentiation competence in air-liquid interface (ALI) cultures. The apical secretions of cultured NHTBE cells were characterized by immunoblotting, Western blotting, or enzyme-linked immunosorbent assay using a variety of antibodies. They contained mucin-like materials as well as lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (SLPI). We found that an EGF concentration of 25 ng/ml, which is commonly used in airway cell cultures, adversely affected growth, mucin production, and morphology of ALI cultures and that RA was essential for mucociliary differentiation. Without RA, the epithelium became squamous and mucin secretions decreased 300- to 900-fold. In contrast, secretion of lysozyme, lactoferrin, and SLPI was significantly increased in RA-depleted cultures. Cells of passage 2 (P-2) through P-4 remained competent to differentiate into mucous and ciliated cells when grown in ALI cultures. However, mucin secretion and ciliagenesis decreased in P-3 and P-4 cell cultures and P-3 but not P-4 cell cultures exhibited bioelectric properties characteristic of airway epithelium. We concluded that P-2 and P-3 NHTBE cell cultures retain many important features of normal airway epithelium. This enables one to conduct many studies of airway cell biology with a greatly expanded (6,000-fold) cell pool.

Bronchi↗
PubMed2000

Secretory differentiation of serially passaged normal human nasal epithelial cells by retinoic acid: expression of mucin and lysozyme.

The purpose of this study was to subculture normal human nasal epithelial (NHNE) cells without compromising their ability to differentiate into secretory and ciliated cells and to study the effect of retinoic acid on mucous and serous secretions in passaged cells and to compare the expression of mucin and lysozyme in cultured cells with those in in vivo nasal epithelium. The subcultured cells were tested after every passage for secretory differentiation in air-liquid interface cultures. The cultured NHNE cells secreted mucin and lysozyme. The cells became squamous and mucin secretion decreased when retinoic acid was deleted from the culture media. Cells from passage 1 through passage 2 remained able to differentiate into mucous or squamous cells. Mucin gene 4 (MUC4), MUC5AC, MUC7, MUC8, and lysozyme messenger RNAs were expressed in passage 2 NHNE cells. In conclusion, passage 2 NHNE cell cultures retain features of normal epithelium and are suitable for many studies of upper airway cell biology.

Cell Differentiation↗
PubMed1995

The in vitro growth and serial passage of RA 27/3 rubella vaccine virus in cord blood mononuclear leukocytes from normal babies.

Because of our inability to isolate rubella virus from peripheral blood lymphocytes from patients with chronic symptoms after rubella immunization we performed a series of experiments with cord blood mononuclear cells (CBL) to determine whether and to what extent these cells supported the growth of rubella virus in vitro. CBL were stimulated with phytohemagglutinin for 48-72 h, washed, and inoculated with 1000 tissue culture 50% infection dose of rubella virus and placed in RPMI 1640 medium with 10% fetal bovine serum and IL-2. Approximately every 9 d the volume was halved, and new CBL were added. Aliquots at 2- to 9-d intervals were obtained and quantitatively cultured in African green monkey tissue culture. Rubella virus growth was determined by echovirus 11 challenge. Controls consisted of rubella virus in RPMI 1640 medium. Rubella virus persisted in CBL culture longer than in control medium in seven of 11 experiments, and in three instances there was evidence of productive infection.

Animals↗