In Vitro最新文献

A methodology for obtaining reproducible in vitro induction of the synthesis of the acute phase reactant serum amyloid P-component (SAP) by purified mouse hepatocytes was established. Optimal hepatocyte culture conditions for the induction and synthesis of SAP required certain hormones, a substratum for cell attachment, and activated macrophages. Leibowitz L15 medium had to be supplemented with dexamethasone, indomethacin, insulin, glucose, and fetal bovine serum. Purified mouse IL 1 could substitute for activated macrophages in the induction of SAP. Hepatocytes were allowed to adhere to a collagen matrix to enhance both cell viability and SAP synthesis induced by IL 1. Elicited macrophages cultured with hepatocytes were capable of augmenting SAP synthesis in the presence of IL 1.

Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco's modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a "hill and valley" pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-35]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, alpha-actin. The identity of alpha-actin is confirmed by analysis of NH2-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of alpha-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation.

Tissue culture offers a model system with which to study the endocrine-mediated growth, differentiation, and metabolic activities of the endometrium. We have established and continue to maintain monolayer cultures of normal human endometrial epithelial cells from each phase of the menstrual cycle. At present, eight proliferative, two secretory, and two menstrual phase cultures have been established. These have been passed at least three times. One proliferative phase culture has been growing for 18 mo, and passed 10 times. Colonies of epithelioid cells as well as single cells appear in the cultures within 2 to 8 h of initial culture and maintain this appearance throughout long-term growth. The cells are periodic acid Schiff positive for carbohydrates and positive for keratin, an immunochemical marker for epithelial tissues. Studies comparing the ultrastructure of the cultures with fresh endometrial tissue revealed morphologic features common to both, including prominent nucleoli, Golgi, mitochondria-rough endoplasmic reticulum complexes, and abundant glycogen. The cells are not tumorigenic in the nude mouse and do not form colonies on soft agarose, confirming the nonneoplastic identity of the cells.

A human hepatocellular carcinoma cell line (FOCUS--Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular origin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, alpha 1-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectable in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and its contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous injection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.

The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg2+ to Mn2+ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV.

Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r = 0.96, P less than 0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.

Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of the Eco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes of Mycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants, Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, and Acholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas.

Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 microM glucosamine. A higher concentration of 35SO4, 1000 microM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 microgram/ml increased the rate of secretion twofold, whereas 0.1 to 100 micrograms/ml of hydrocortisone and 0.1 to 100 micrograms/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10(-5) M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10(-9) M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2 M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistinguishable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.

Human myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue.

The main obstacle to establishing pure normal human melanocytes in vitro is contamination of the cultures by fibroblasts. The obstacle can be overcome by selective destruction of fibroblasts with geneticin ( G418 sulfate). Treatment of mixed cultures with this drug at a concentration of 100 micrograms/ml for two days results in pure cultures of normal human melanocytes.