Days Of Subculture Research Articles

A procedure for long-term culture of oligodendrocytes

A procedure for long-term culture of oligodendrocytes is described, the starting material being 20-day-old primary mixed cultures of newborn rat brain. Cells were first incubated in a serum-free medium for 48 h before they were subcultured on poly- l-lysine coated plastic dishes. After this treatment, the oligodendrocytes developed well in Waymouth medium containing 10% (v/v) calf serum, while most of the astrocytes died. At 13 days in subculture more than 90% of the cells were identified as oligodendrocytes; the criteria for oligodendrocytes were based on their immunoreactivity to antisera against W1 Wolfgram protein, myelin basic proteins and the synthetic C-terminal hexapeptide of the major myelin proteolipid. At 13 and 19 days astrocytes were present, 7% and 20% respectively. The culture system described here may be useful to study the biochemical and immunological aspects of the oligodendrocytes.

Absolute rates of sterol synthesis estimated from [3H]water for bovine lens epithelial cells in culture.

All cells of the avascular ocular lens derive from a monolayer of epithelial cells located on only the anterior surface of this organ. The source of the cholesterol required for the growth and division of these cells was studied by using cultures of bovine lens epithelial cells. Cells were in active growth during the third to fourth day of subculture following seeding. Absolute rates of cholesterol synthesis were estimated for the cultured cells from incorporation of [3H]water. Rates were estimated on the assumption that 0.81 atoms of 3H of [3H]water were incorporated into cholesterol per carbon atom of cholesterol, a situation where all of the NADPH would be generated by oxidative enzymatic processes. We tested this assumption by measuring the changes in sterol mass per dish of cells grown in lipoprotein-deficient media over day 3 to 4 of subculture and by simultaneously measuring the rates of incorporation of [3H]water into sterols during this period. In this situation, the increases in sterol mass should be attributable solely to de novo sterol synthesis. We calculated that an average of 0.79 atoms of 3H of [3H]water were incorporated by these cells into cholesterol per carbon atom of cholesterol. Sterol synthesis was only modestly decreased (about 30%) when the cells were cultured in media prepared with whole calf serum. Growth rates of the cells were also little affected by the absence of lipoproteins. In spite of the capacity to furnish its sterol requirements by de novo synthesis, the lens epithelial cells readily degraded 125I-labeled bovine LDL, and LDL greatly decreased sterol synthesis when added to the media at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The Degradation of 2,4-Dichlorophenoxyacetic Acid by Maize Cells in Suspension Culture

Summary About 40 per cent of [1-14C]2,4-D was degraded by maize cells during the first 3–4 days of subculture. A comparison of the metabolism of [1-14C]2,4-D and [2-14C]2,4-D led to the conclusion that the first step in 2,4-D degradation is the splitting off of the side chain. The rate of degradation increased several times during the first 15 h of subculture and then declined concomitantly with a decrease of intracellular 2,4-D content. An estimation of the relative rate of degradation (rate of degradation: intracellular 2,4-D content) revealed it to be diminished when 2,4-D content in the cells decreased under the influence of some experimental factors. The presence of two pools of intracellular 2,4-D — one fast-effluxing and the other saturable slowly-effluxing is postulated on the ground of efflux analysis. It was concluded that only 2,4-D in the fast-effluxing pool is available for degradation. The influence exerted by some parameters (initial 2,4-D concentration, pH of the medium, suspension density, physiological state of the cells) on the rate of 2,4-D degradation depends on the changes of intracellular 2,4-D content, compartmentation of 2,4-D and metabolic activity.

Cultured rabbit vesical smooth muscle cells for lining of dissolvable synthetic prosthesis

For prosthetic use in genitourinary operations we investigated the feasibility of producing grafts from cultured smooth muscle cells on absorbable synthetic biomaterial. Biopsies of bladder wall muscle without mucosa were obtained from 20 rabbits for culture of smooth muscle cells; cultured cells were examined by light and electron microscopy. Those cells were grown in subculture on a microfiber mesh carrier woven from 35-denier polyglycolic acid fibers. As shown by scanning electron microscopy (SEM), complete coverage of the synthetic fabric was achieved after ten to fourteen days of subculture.

Growth characteristics and chromosomal behavior of cell suspension lines established from pollen callus of peony.

Six cell suspension lines (Kp9, Kp10, Kp20, Kp42, Tp1 and Tp4) established from pollen calli of two Paeonia cultivars have been studied with special reference to their growth characteristics and chromosomal behavior. They reached stationary phase after about 14 days of subculture showing about a 6- to 10-fold increase in packed cell volume, regardless of their ploidy levels. The mitotic index in each line showed a maximum value of between 6-8% after about 5 days of subculture.Haploid cell lines of Kp9, Kp20 and Tp1 maintained haploid chromosome numbers (n=5) at high values (more than 90%) even after 620 days of subculture. However, the Kp42 line changed from the original haploid to the mixoploid by an increase in diploid cells during subculture. The diploid Kp10 and the mixoploid Tp4 lines retained their characteristic chromosome number throughout subculture. In the mixoploid Kp42 and the Tp4 lines, the cells having 5 or 10 diplochromosomes resulting from endoreduplication in a haploid or a diploid nucleus were occasionally found.

Plant Regeneration from Callus Culture in Wheat 1

Plant regeneration was investigated from embryo-callus culture in bread wheat, Triticum aestivurn L. Immature embryos of cultivar ‘Maris Huntsman’ were cultured on half-strength Murashlge and Skoog's medium (half-MS) supplemented with 0.5 mg 2,4-D[(2,4-dichlorophenoxy) acetic acid], 3.2 mg IAA (indole-3-acetic acid) and 1.0 mg kinetin [6-(furfurylamino) purlne]/liter or without kinetin. On medium with 2,4-D, IAA and kinetin, 77% of the embryos produced a visible callus within 2 weeks. On medium without kinetin, all embryos produced a callus. Calll were cloned and maintained for over 900 days by subculturing at 50 to 60 days interval on half-MS medium with 0.5 mg 2,4-D/liter but regeneration ceased after 680 days. Calli showed organogenesis within 120 days of subculture on half-MS medium with 0.5 mg 2,4-D and 1.0 mg zeatin [6-(4-hydroxy-3-methyl but-2 enylamlno) amino purine]/liter. Plantlets originated from callus by differentiation into shoot-primordia and bipolar structures. Plantlets produced roots on half-MS medium without growth regulators; however, 1.0 mg naphthalene acetic acld/llter in the medium promoted rooting. Regenerated plants included variants for plant height, stem thickness, leaf size, spike shape, pollen fertility and seed set.

Influence of cell density on desensitization of the thyroid cell cyclic adenosine 3',5'-monophosphate response to thyrotropin stimulation.

Stimulation of cultured thyroid cells with TSH produces an acute increase in cAMP content (15–30 min maximum) followed by a decrease over 24 h representing refractoriness to further TSH stimulation. With continued exposure of thyroid cell subcultures to TSH for greater than 4 days, escape from desensitization occurred in that cAMP levels returned to the initial maximum (15–30 min) values. This increase in cAMP levels was unrelated to an increase in cell number. In addition, subculture of thyroid cells for between 1 and 6 days in the absence of TSH followed by acute (30 min) TSH stimulation revealed a progressive decrease in the maximum cAMP levels attained (50% of day 1 values after 4 and 6 days of subculture). The cAMP response to a 24-h period of TSH stimulation was examined in groups of cells after different durations of subculture without TSH. With TSH stimulation on the first day of subculture, as expected, cAMP levels increased markedly after 30 min, declined by 6 h, and remained suppressed for up t...

Uptake of 2,4-Dichlorophenoxyacetic Acid by Suspension-cultured Maize Cells

The uptake of 2,4-D-[1- 14 C] by maize cells was studied in relation to 2,4-D concentration, density of suspension, growth phase of subculture and p H of the medium. The uptake was estimated by the equilibrium 2,4-D content in the cells (C t ) and by the relative rate of the approach of cell 2,4-D content to C t (k) which was shown to be constant during the uptake. An increase in suspension density caused a decrease in C t and an enhancement of (k) . The exponential phase cells (4th day of subculture) took up 2,4-D more actively than the stationary phase cells just transferred to the fresh medium. This activation of uptake can only partly be conditioned by the acidification of the medium during the subculture. An increase in 2,4-D concentration in the medium from 1 mg/l to 5-10 mg/l caused an enhancement of k in the initial culture and its decrease in the 4th day culture. Only part of absorbed 2,4-D could be washed out by three successive 1 h incubations of the cells with the medium without 2,4-D. The non-removed (or very slowly removed) 2,4-D was equal near to 70 % of total absorbed 2,4-D in the cells of the 4th day culture and 20 % in the cells of the initial culture.

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

A saline extract was prepared from Drosophila eggs. When diluted to a concentration of 1% with Drosophila tissue culture medium, it did not support growth of cells from the Drosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time four Drosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonic Drosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells.

Effects of prostaglandin E 1 and isoproterenol on cyclic AMP content of human fibroblasts modified by time and cell density in subculture

In confluent cultures of “young” (< 30 generations) human fibroblasts, maximally effective concentrations of prostaglandin E 1 (5.6 μM) and isoproterenol (2 μM) increased cyclic AMP content several hundred-fold and approximately 30-fold, respectively. On the first day after initiation of cultures at either low (approx. 3 · 10 5 cells) or high (approx. s · 10 6 cells) cell density the magnitude of the isoproterenol effect was similar to that in confluent cultures. It increased during the next few days, reaching a maximum around day 2–3, and then declined. On any day during the period of subculture, the magnitude of the isoproterenol effect was inversely related to cell density. Alterations in response to prostaglandin E 1 as a function of time in subculture or cell density were less dramatic. The effects of prostaglandin E 1 were, however, smaller at some point during the first few days of subculture than after day 7, and when effects of prostaglandin E 1 were minimal, those of isoproterenol were maximal and approached those of prostaglandin E 1. On any day of subculture, cells in cultures of higher density tended to accumulate more cyclic AMP in response to prostaglandin E 1 than did those in low density cultures. The effects of prostaglandin E 1 and isoproterenol on cyclic AMP content were qualitatively similar in “young” and in “old” (> 60 generations in culture) human fibroblasts although the changes associated with duration of subculture and cell density tended to be less marked with “old” cells. In the “young” fibroblasts responsiveness to isoproterenol and prostaglandin E 1 appears to correlate with cell morphology and with the fractional rate of growth in subcultures. It is suggested that the capacities of the fibroblasts to respond to these two agents may be altered independently during growth of human fibroblasts.