Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures

The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells.     

The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells.

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.     

Cell cultures of fetal rat brain : Growth and marker enzyme development

Growth and enzyme development in cell cultures of fetal rat brain were influenced by type of growth medium, cell density, and age of fetal tissue source. Cells grew better in one medium (DMEM), but the other (F12G) enhanced development of choline acetyltransferase activity. One type of growth medium (DMEM) lost efficacy 2 weeks after preparation of complete medium. Cell division rate was density dependent, and choline acetyltransferase development was related to time in culture and cell concentration. Some results suggested division of choline acetyltransferase producing cells. Differences in age of tissue source resulted primarily in differences in growth: cultures of 21 day fetal cells developed more protein per 10⁶ cells inoculated than cultures of cells from younger animals; there was little difference in enzyme activity per culture. Conditions may be controlled such that fetal rat brain cells will grow and express differentiated functions in culture in a predictable manner.     

Effects of plating density and age in culture on growth and cell division of neonatal rat heart primary cultures

Summary The cell-type composition of the initial cell population from protease-dispersed neonatal rat heart tissue has been evaluated using time lapse photography and identification of cell type-specific functions. The effects of two commonly employed plating densities on growth and cell division of the two major cell types were examined. Total protein synthesis rates were not affected by plating density but did change with age in culture. Maximum protein synthesis rates were observed during the period of maximum cell division and cell growth (increase in total cell protein), which was from 24 h in culture to the 4th d in culture. After 6 d in culture, synthesis rates for total proteins remained constant for at least 2 wk. Sizing of cells by Coulter counter analysis indicated that essentially all the cells were increasing in size with age in culture. Measurements of cell numbers and rate of DNA synthesis indicated that the extent of cell division was dependent on plating density. Cells disaggregated from neonatal rat hearts consisted of approximately 75% muslce cells and 25% nonmuscle cells. This composition approximates the cell-type composition of the intact neonatal rat heart. In high density cultures there is little cell division and the relative proportionsof the cell types are preserved with time in culture. In low density cultures, proliferation of nonmuscle cells is a significant process and the composition of the cell population changes drastically during the first 2 to 3 d in culture. These results suggest that the low plating density used by many researchers may limit correlation of data derived from such cultures with the physiological state. It also indicates that plating densities should be given in published accounts for comparisons to be made with results from other laboratories. This work was supported in part by U.S. Public Health Service Grant HL10018 and The Pennsylvania State University Agricultural Experiment Station and was authorized for publication as Paper 5490 in the journal series of the Pennsylvania Agricultural Experiment Station.     

The role of cell-cell contact in "contact" inhibition of cell division: a review and new evidence

The term “contact inhibition of cell division” was borrowed from “contact inhibition of cell movement.” We prefer the term “postconfluence inhibition of cell division” as being more operational and less mechanistically biased; it is operationally defined as a pronounced depression of the mitotic rate in a postconfluent culture which displays a stationary density despite periodic nutrient renewal, the inhibition being locally reversibly by removal of the adjacent cells. The mechanism of postconfluence inhibition is of considerable interest because of the inverse correlation between postconfluence inhibition and the tumorigenicity of a number of cell lines. Several hypotheses, involving direct cell-to-cell contacts or locally restricted diffusion gradiens, could explain postconfluence inhibition. With the goal of discriminating among these hypotheses, time-lapse films were taken of carefully regulated, perfused cultures of 3T3 mouse cells, in which the transition from rapid growth to the stationary phase was recorded. Measurements of cell-to-cell contact, local cell density, and generation times were made on an individual cell level and analyzed with the aid of a computer. We observed that all-around cell-cell contact or a high local cell density present throughout G1 often did not produce immediate inhibition of cell division. We conclude that either (i) simple visible cell-cell contacts or a high local cell density are not the direct cause of postconfluence inhibition of cell division, or (ii) their effects often do not inhibit cell division until after a delay of about one cell generation time. Such a delay may be partly responsible for the 50% overshoot past the stationary density that we observed in 3T3 cultures.     

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

Evidence for contact stimulation of growth of homologous cells in superinoculated cultures of chick embryo cells

The secondary cultures of chick embryo cells were suspended and transferred to homologous cell cultures. Cell adhesion and proliferation were studied in these superinoculated cultures. It was shown that added cells soon adhered to the underlying cell layer which results in a prompt increase in culture density followed by the activation of DNA synthesis and cell division. Stimulation of cell proliferation involved both cell subpopulations composing the superinoculated culture: cells seeded on the built-up cell layer and cells of the layer. The contact nature of added cell mitogenic action on overlaid cell proliferation was evidenced. The cell system described can be used to investigate the adhesive properties of the cell layer apical surface, the relationship between cell growth rate and culture density, and the contact stimulation of cell proliferation.     

Blue light delays commitment to cell division in Chlamydomonas reinhardtii

In this study, we describe the effect of red and blue light on the timing of commitment to cell division in Chlamydomonas reinhardtii. The time point and cell size after which cells can complete their cell cycle with one division round were determined for cultures that were exposed to various red and blue light periods. We show that the commitment point of cells grown in blue light is shifted to a later time point and a larger cell size, when compared with cells grown in red light. This shift was reduced when cultures were exposed to shorter blue light periods. Furthermore, this shift occurred only when exposure to blue light started before the cells attained a particular size. We conclude that the critical cell size for cell division, which is the cell size at which commitment to cell division is attained, is dependent on spectral composition.     

Acetylcholinesterase expression and changes in the electrical excitability of neuroblastoma cells during their morphological differentiation induced by an increase in the pH of the medium

Inhibition of cell division and outgrowth of neurites with average rate of 31.5 +/- 4.4 micrometers per hour were observed in neuroblastoma cultures of the Neuro 2a clonal line 24 hours after the increase in the culture medium pH from 7.4 to 8.2. The total neurite length per one cell was about 298 +/- 36 micron in average by the 9-10th days of treatment. Simultaneously, a gradual enhancement of acetylcholinesterase cytochemical appearance took place attaining its maximum level by the same time. The peak sodium conductance, taken as a measure of sodium tetrodotoxin-sensitive potential-dependent channel density, was the same both in nondifferentiated cells grown in suspension or monolayer cultures, and in morphologically differentiated ones. The data lead to a conclusion that biochemical (acetylcholinesterase probe) and electrophysiological (sodium channel density) signs can express independently of morphological differentiation.     

Synthesis and Structure of Caulobacter crescentus Flagella

During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.     

Fluctuations in buoyant density during the cell cycle of Escherichia coli K12: significance for the preparation of synchronous cultures by age selection.

The buoyant densities of Escherichia coli K12 were investigated by isopycnic centrifugation in gradients of colloidal silica (Ludox) and polyvinylpyrrolidone. Bacteria from an exponential culture in a defined medium supplemented with hydrolysed casein banded at densities between 1-060 and 1-115 g ml-1; the mean density was 1-081 g ml-1. At the higher densities, two populations of cells were present: smaller cells were approximately twice as numerous as, and half the modal volume of, the population of larger cells. A homogeneous population of cells of intermediate volume equilibrated in the least dense region of the density band. Synchronous cultures were established by inoculating cells selected from the most or least dense regions of the band into spent growth medium. The results are consistent with a fluctuation between maximal density at cell birth and division, and minimal density near the middle of the cell cycle. In synchronous cultures prepared by continuous-flow age selection, the first division occurred after a period that was significantly shorter than the length of subsequent cell cycles. Cells selected by this procedure were of similar mean density to those in the exponential culture but were more homogeneous with respect to size. The possibility that the smallest (and densest) cells in an exponential culture are retained in the rotor, and are thus excluded from the synchronous culture, is discussed.     

Autogamy in Paramecium cell cycle stage-specific commitment to meiosis

Autogamy is a process of meiosis and fertilization which takes place in unpaired Paramecium cells, and which is triggered by starvation. This study examines the consequences of nutritional down-shift at various points within the cell cycle on the occurrence of autogamy. It shows that cells become committed to autogamy in a two-step process. An initial point of commitment to autogamy occurs about 100 min prior to the median time of cell division (cell cycle duration, 330 min). Cells which have become committed to autogamy initiate meiosis following the next fission, others complete another vegetative cell cycle before undergoing meiosis. Treatments that perturb the cell cycle and displace the point of commitment to division also displace the point of initial commitment to autogamy to the same extent.The initial commitment to autogamy can be reversed by refeeding. The second, final, point of commitment to autogamy occurs about 30 min after the fission, immediately prior to initiation of meiosis, and coincides with the beginning of meiosis. If cells are refed at this point, or at later stages, autogamy continues.Autogamy is not well synchronized either in naturally starved cultures or in those subjected to abrupt nutritional down-shift. This is a consequence of the cell cycle stage dependence of entry into autogamy. Autogamy occurs synchronously in samples of dividers selected from asynchronous cultures 2 or more hours after nutritional down-shift. The timing of the events of conjugation and autogamy coincide when the pre-autogamous fission is aligned temporally with the initial contact of mating cells.     

Buoyant density constancy of Schizosaccharomyces pombe cells.

Buoyant densities of cells from exponentially growing cultures of the fission yeast Schizosaccharomyces pombe 972h- with division rates from 0.14 to 0.5 per h were determined by equilibrium centrifugation in Percoll gradients. Buoyant densities were independent of growth rate, with an average value (+/- standard error) of 1.0945 (+/- 0.00037) g/ml. When cells from these cultures were separated by size, mean cell volumes were independent of buoyant density, indicating that buoyant densities also were independent of cell age during the division cycle. These results support the suggestion that most or all kinds of cells that divide by equatorial fission may have similar, evolutionarily conserved mechanisms for regulation of buoyant density.     

Reduction of Selenite and Detoxification of Elemental Selenium by the Phototrophic Bacterium Rhodospirillum rubrum

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1.5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

Amino Acid Pools and Metabolism During the Cell Division Cycle of Arginine-Grown Candida utilis

Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed.     

The Influence of Mature Tissue 2

Observations have been made on rates of increase in total numberof cells and on rates of division in isolated pea roots grownin sterile conditions in one series over a period of 9 daysand in another started from tips of different lengths. It isshown that when cultures are started from tips of 6.0 mm. excisedfrom germinated seeds the rate of increase in cell numbers increaseswith time until the fifth day and then decreases to the ninthday. It is further shown that when cultures are started fromtips of 3.0, 6.0, and 10.0 mm., the initial lag phase is longer,the peak rate in division occurs later, and the peak value tendsto be greater the shorter the initial tip. These observationsare taken to indicate that division in the meristem dependspartly on the synthesis of appropriate metabolites in the meristemand partly on a supply of metabolites from mature regions ofthe root.     

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

Cell cycle of Chlorella vulgaris can deviate from the synchronous binary division model

Conditioned medium from high density Chlorella vugaris cultures was freeze dried extracted into ethanol. The ethanol was volatilized and the resulting powder then introduced with C. vulgaris photoautotrophic cultures which underwent asynchronous DNA replication forming cells with two, three, four, and six autospores instead of 2 n characteristic of synchronous DNA replication. These cells were blocked at the division stage of the cell cycle.