In Vitro最新文献

The Mishell-Dutton culture system for in vitro primary antibody response of mouse spleen cells was used to optimize the amino acid composition of RPMI 1640 media. Each of the 20 amino acids was tested over a broad range of concentrations always leaving the remaining 19 amino acids unaltered (i.e. at the formula recommended concentration). In several instances, higher plaque-forming cell responses were obtained with an amino acid concentration that was either higher or lower than that recommended: (a) the optimum concentration for valine, glutamine, and lysine lies considerably above the recommended one, (b) the optimum concentration for leucine as well as for several other amino acids lies below the recommended concentration, and (c) the optimum concentration for arginine corresponds exactly to the recommended concentration. The second round of optimization, i.e. combining of two conditions that individually yielded an improved response often caused a decrease of response. The possibility is discussed that for an optimal response a ratio of two or several amino acids rather than the absolute concentration of any one amino acid is of importance.

In vitro growth of rat atriocaval epithelial tumor cells (ACT-1) was enhanced by the inclusion of xenogeneic mouse adherent peritoneal exudate cells (PECs) in a two-layer soft agar system. A linear relationship was found between the number of cells plated and the number of colonies when ACT-1 tumor cells were plated at plating densities of between 1 and 5 X 10(5) cell/60 mm plate (r = 0.9, P less than 0.001). Inclusion of irradiated PECs in the bioassay for tumor stem cells resulted in a two and a half-fold increase in colony formation in three separate experiments (P less than 0.001).

We have carried out systematic studies to optimize and standardize methodology to isolate and culture the adult rat ventricular cardiac muscle cell. Four hearts were perfused simultaneously with a calcium-free medium containing collagenase. The ventricular tissue was then minced and further digested to liberate individual cells. Approximately 16 million rod-shaped muscle cells were obtained. The plating efficiency has been greatly improved by culturing the cells in a conditioned medium prepared from a rabbit corneal cell line. This medium also contained added fetal bovine serum, essential and nonessential amino acids, vitamins, insulin, transferrin, and 25 trace minerals. The culture flasks were precoated with rat-tail collagen. Fibroblast contamination was virtually eliminated by including cytosine arabinoside in the medium during the first 7 d of culture. After this time the cells could be cultured in the absence of serum in a chemically defined medium composed of MEM, vitamins, nonessential amino acids, and trace minerals. They continued to contract spontaneously and do well in this medium for at least 3 d thereafter. This improved methodology resulted in a reproducible culture system with improved plating efficiency. It provided a new and unique system to study the structure and function of the adult mammalian ventricular cardiac muscle cell.

In a study of a diverse set of human tumor cell lines previously shown to all have a defect in methionine metabolism (Stern, P. H., Wallace, C.D. and Hoffman, R.M., J. Cellular Physiology 119, 29-34, 1984), we demonstrate in this report that all have enhanced overall rates of transmethylation compared to normal human fibroblasts. Transmethylation rates were measured by blocking S-adenosylhomocysteine hydrolase and measuring the AdoHcy which accumulates as a result of transmethylation. The enhanced transmethylation rates may be the basis of the above-mentioned defects in methionine metabolism previously reported in human tumor cells, including the basis of the inability of the majority of the tumor cells to grow when methionine is replaced by homocysteine. The excess and unbalanced tRNA methylation observed for the last 25 years in many types of cancer may be at least in part explained by our results of elevated rates of overall transmethylation in cancer cells. The alteration of such a fundamental process as transmethylation in cancer may be indicative of its importance in the oncogenic process.

The concentration of Ca++ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca++ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of alpha-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca++ to less than 0.08 mM. The reduction of Ca++ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca++ on growth and differentiation were specific (Mg++ and Mn++ variations were without effect) and reversible and in many respects resembled Ca++ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca++-induced coupling and uncoupling of proliferation and the program of differentiation, we proposed that Ca++ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu.

Primary cultures of beating myocardial cells were obtained from 5-d-old offspring of trained (T) and untrained (UT), pregnant, Sprague-Dawley rats. The myocardial cells from the T and UT groups were evaluated for their beating responses to three cardioactive drugs: verapamil (V), isoproterenol (ISO), and propranolol (PRO). The myocardial cell cultures from the UT group showed complete loss of beating when the calcium (Ca++) antagonist, V, was added to the cultures for 1 h or more; the T group was able to show some beating at comparable concentrations and durations of exposure with V. The beta agonist, ISO, markedly stimulated the beating rate of both the T and UT groups, but the beating rates were higher in the UT group at comparable concentrations and durations of exposure than with the T group. When the cultures were pretreated with the beta blocker, PRO, before treatment with ISO, a concentration inhibitory effect on the beating rate was observed in both groups. However, the T cultures were more sensitive to the inhibitory effects of PRO. These results demonstrate that primary cultures of rat myocardial cells isolated from the offspring of trained and untrained pregnant rats show differential beating responses to three well-known cardioactive drugs.

Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolize N-2-acetylaminofluorene to 2-aminofluorene and N-hydroxy-acetylaminofluorene, products that transform hamster embryo cells. Large amounts of N-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels of N-hydroxy-acetylaminofluorene were formed after incubation of N-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested with N-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system.

Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO2 of approximately 142 mm Hg, cultures developed in a pO2 of approximately 30 mm Hg, close to the normal fetal arterial pO2, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO2 approximately 30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO2. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung.

Primary cultures of rat myocytes were deprived of oxygen (approximately 5 mm Hg, pO2) for 2 h in the presence or absence of calcium and were subsequently incubated for different time periods under normoxic (approximately 120 mm Hg, pO2) conditions. Myocyte calcium content was determined at the end of the oxygen-free period and after repletion of oxygen. Lactate dehydrogenase (LDH) release from the cells into the medium was used as an index of cell injury. Cultures deprived of oxygen in the absence of calcium showed a significant decrease in myocyte calcium content at the end of the oxygen-free period. However, the calcium content of myocyte cultures deprived of oxygen in the presence of calcium did not change significantly. The enzyme release of both groups of oxygen-deprived cultures was similar to that of controls. A progressive increase in myocyte calcium content was observed 5, 10, 15, and 30 min after repletion of oxygen in both groups. Reoxygenation of cultures subjected to hypoxia in the presence of calcium caused minimal LDH leakage. However, major enzyme release was observed 30 min after oxygen repletion in cultures previously subjected to hypoxia in a calcium-free environment. The present study shows that reoxygenation injury is more severe in myocyte cultures previously deprived of both oxygen and calcium than in cultures deprived of oxygen in the presence of calcium.

Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride). When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated the cell growth, and the effect was more pronounced with the ferric chloride solution.