Stationary Cell Density Research Articles

Reconsidering Dogmas about the Growth of Bacterial Populations.

The growth of bacterial populations has been described as a dynamic process of continuous reproduction and cell death. However, this is far from the reality. In a well fed, growing bacterial population, the stationary phase inevitably occurs, and it is not due to accumulated toxins or cell death. A population spends the most time in the stationary phase, where the phenotype of the cells alters from the proliferating ones, and only the colony forming unit (CFU) decreases after a while, not the total cell concentration. A bacterial population can be considered as a virtual tissue as a result of a specific differentiation process, in which the exponential-phase cells develop to stationary-phase cells and eventually reach the unculturable form. The richness of the nutrient had no effect on growth rate or on stationary cell density. The generation time seems not to be a constant value, but it depended on the concentration of the starter cultures. Inoculations with serial dilutions of stationary populations reveal a so-called minimal stationary cell concentration (MSCC) point, up to which the cell concentrations remain constant upon dilutions; that seems to be universal among unicellular organisms.

A repertoire of repulsive Keller–Segel models with logarithmic sensitivity: Derivation, traveling waves, and quasi‐stationary dynamics

In this paper, we show how the chemotactic model introduced in Alejo and López (2021), which accounts for a chemical production–degradation operator of Hamilton–Jacobi type involving first‐ and second‐order derivatives of the logarithm of the cell concentration, namely, with , can be formally reduced to a repulsive Keller–Segel model with logarithmic sensitivity whenever the chemotactic parameters are appropriately chosen and the cell concentration keeps strictly positive. In this way, some explicit solutions (namely, traveling waves and stationary cell density profiles) of the former system can be transferred to a number of variants of the the latter by means of an adequate change of variables.

Evolutionary engineering of Escherichia coli for improved anaerobic growth in minimal medium accelerated lactate production

Anaerobic fermentation is a favorable process for microbial production of bulk chemicals like ethanol and organic acids. Low productivity is the bottleneck of several anaerobic processes which has significant impact on the technique competitiveness of production strain. Improving growth rate of production strain can speed up the total production cycle and may finally increase productivity of anaerobic processes. In this work, evolutionary engineering of wild-type strain Escherichia coli W3110 was adopted to improve anaerobic growth in mineral medium. Significant increases in exponential growth rate and stationary cell density were achieved in evolved strain WE269, and a 96.5% increase in lactate productivity has also been observed in batch fermentation of this strain with M9 minimal medium. Then, an engineered strain for lactate production (BW100) was constructed by using WE269 as a platform and 98.3g/L lactate (with an optical purity of D-lactate above 95%) was produced in a 5-L bioreactor after 48h with a productivity of 2.05g/(L·h). Finally, preliminary investigation demonstrated that mutation in sucD (sucD M245I) (encoding succinyl-CoA synthetase); ilvG (ilvG Δ1bp) (encoding acetolactate synthase 2 catalytic subunit), and rpoB (rpoB T1037P) (encoding RNA polymerase β subunit) significantly improved anaerobic growth of E. coli. Double-gene mutation in ilvG and sucD resumed most of the growth potential of evolved strain WE269. This work suggested that improving anaerobic growth of production host can increase productivity of organic acids like lactate, and specific mutation-enabled improved growth may also be applied to metabolic engineering for production of other bulk chemicals.

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation.

BackgroundDictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.ResultsWe find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a KD of 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Ki of 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA.ConclusionTogether, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA- cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.

A continuum mathematical model of endothelial layer maintenance and senescence

BackgroundThe monolayer of endothelial cells (ECs) lining the inner wall of blood vessels deteriorates as a person ages due to a complex interplay of a variety of causes including cell death arising from shear stress of blood flow and cellular oxidative stress, cellular senescence, and decreased rate of replacement of dead ECs by progenitor stem cells.ResultsA continuum mathematical model is developed to describe the dynamics of large EC populations of the endothelium using a system of differential equations for the number densities of cells of different generations starting from endothelial progenitors to senescent cells, as well as the densities of dead cells and the holes created upon clearing dead cells. Aging of cells is manifested in three ways, namely, losing the ability to divide when the Hayflick limit of 50 generations is reached, decreasing replication rate parameters and increasing death rate parameters as cells divide; due to the dependence of these rate parameters on cell generation, the model predicts a narrow distribution of cell densities peaking at a particular cell generation. As the chronological age of a person advances, the peak of the distribution – corresponding to the age of the endothelium – moves towards senescence correspondingly. However, computer simulations also demonstrate that sustained and enhanced stem cell homing can halt the aging process of the endothelium by maintaining a stationary cell density distribution that peaks well before the Hayflick limit. The healing rates of damaged endothelia for young, middle-aged, and old persons are compared and are found to be particularly sensitive to the stem cell homing parameter.ConclusionThe proposed model describes the aging of the endothelium as being driven by cellular senescence, with a rate that does not necessarily correspond to the chronological aging of a person. It is shown that the age of the endothelium depends sensitively on the homing rates of EC progenitor cells.

Raised level of cyclin dependent kinase A after prolonged suspension culture of Nicotiana plumbaginifolia is associated with more rapid growth and division, diminished cytoskeleton and lost capacity for regeneration: implications for instability of cultured plant cells

A long maintained suspension culture of Nicotiana plumbaginifolia, compared with a newer culture, had increased levels of cyclin dependent kinase A (CDKA;1) a halved cell doubling time and twice the stationary cell density. The newer culture downregulated CDKA protein levels by 75% during the transition to stationary phase and, in mixtures of natural and synthetic auxins with cytokinins, developed shoot tissue that could regenerate into plants, while the older cell line developed only callus. The older cell line retained some posttranslational regulation of CDK catalytic activity that was indicated by lower CDK enzyme activity in stationary phase and by a peak profile of CDK enzyme activity at mitosis in synchronous culture. Only the older cell line lacked preprophase band (PPB) microtubules and took 1 h less time after S phase to reach cytokinesis. It is suggested that the raised CDK activity after prolonged culture has multiple effects, including a marked shortening of G1 phase by a universal mechanism of CDK activation of transcription factors for DNA synthesis, and prevention of PPB assembly consistent with earlier observed effects of CDK microinjection, as well as a predicted elevation of mutagenesis by opposition of checkpoint arrest. Proliferation remained in balance with faster growth, which may have been accelerated by CDK activation of transcription factors. It is concluded that cells in which mutations elevate CDK expression and accelerate proliferation will inevitably predominate when populations are propagated by mass transfer. High CDK activity in such cells obstructs plant regeneration by preventing assembly of the PPB cytoskeleton and by countering restriction of cell division to meristems and the termination of proliferation that is required for organ development.

Apoptosis involved in density-dependent regulation of rat fibroblastic 3Y1 cell culture.

When rat 3Y1 fibroblastic cells are cultured toward confluency, the cells go through maximum cell density (overshoot) before reaching post-confluence stationary cell density. After overshooting, a number of floating cells are found in the medium. In a long-term culture, a cyclic change in cell number, an increase after each medium refreshment and subsequent cell loss within a few days has been observed so that the cell populations in the monolayer maintain post-confluence stationary cell density at a constant level. The floating cells excluded trypan blue, but they had no ability to attach to the substrate and to form colonies after being reseeded in fresh medium. They had condensed and uniformly electron-dense chromatin with sharply circumscribed edges. Their DNA contained a laddering pattern in harmony with internucleosomal cleavage. The features were those of apoptosis. When floating cells appeared, apoptotic bodies were also observed in the monolayer. Most of them were found within the cytoplasm of intact cells, suggesting that apoptotic bodies were also faded away from the culture by being rapidly engulfed by neighboring intact cells. These suggest that apoptosis and subsequent detachment from the monolayer or engulfment by neighboring intact cells, in addition to inhibition of cell division, are basic mechanisms on the process of density-dependent regulation in monolayer culture of rat 3Y1 cells.

Integro-differential equations for the self-organisation of liver zones by competitive exclusion of cell-types

AbstractA model is developed for the seif-organisation of zones of enzymatic activity along a liver capillary (hepatic sinusoid) lined with cells of two types, which contain different enzymes and compete for sites on the wall of the sinusoid. An effectively non-local interaction between the cells arises from local consumption of oxygen from blood flowing throug1 the sinusoid, which gives rise to gradients of oxygen concentration in turn influencing rates of division and of death of the two cell-types. The process is modelled by a pair of coupled non-linear integro-differential equations for the cell-densities as functions of time and position along the sinusoid. Existence of a unique, bounded, non-negative solution of the equations is proved, for prescribed initial values. The equations admit infinitely many stationary solutions, but it is shown that all except one are unstable, for any given set of the model parameters. The remaining solution is shown to be asymptotically stable against a large class of perturbations. For certain ranges of the model parameters, the asymptotically stable stationaxy solution has a zonal structure, with cells of one type located entirely upstream of cells of the other type, and with jump discontinuities in the cell densities at a certain distance along the sinusoid. Such sinusoidal zones can account for zones of enzymatic activity observed in the intact liver. Exceptional cases are found for singular choices of model parameters, such that stationary cell-densities cannot be asymptotically stable individually, but together form an asymptotically stable set. Certain mathematical questions are left open, notably the behaviour of large deviations from stationary solutions, and the global stability of such solutions. Possible generalisations of the model are described.

Growth regulation in multilayered cultures of human diploid fibroblasts: the roles of contact, movement and matrix production.

Early subcultures of human embryonic lung fibroblasts are exceptional, as they grow far beyond confluence before growth ceases: the stationary dish may well contain 3-10 monolayer equivalents. Maximal growth rates, however, occur at about one-sixth confluence when doubling times are 15-20 hr; a density at which cell contacts begin to become frequent. The fact that a slowing down of growth is first apparent at such low densities argues against this regulation being due to diffusion effects. Confirmation of the role of short-range or contact interactions in growth regulation comes from an experiment using mixed cultures of fibroblasts: this shows that growth inhibition is not carried by medium-borne influences but depends on short-range (less than 1 mm) interactions. Evidence that cells can escape the effects of such contact interactions and so divide comes from time-lapse studies of dense cultures: there is a burst of motility soon after a fresh-medium change, which is followed by a burst of mitosis approximately 20 hr later. A medium change to conditioned medium supplemented with 10% foetal calf serum leads to neither the burst of motility nor the subsequent burst of mitosis, although this medium is better able to support the growth of sparse cells than is fresh medium. Data are also presented to show that the amount of collagen deposited in superconfluent cultures affects their growth: the stimulation of collagen production with ascorbic acid leads to an unexpectedly low stationary cell density and rather less movement in the culture. This result suggests that the collagen stabilizes cell contacts that are responsible for growth inhibition. The question of why these cells grow more slowly as density increases cannot be answered directly by these experiments; nevertheless, the results suggest that cell contact affects the permeability of the cell membrane to medium.

Density-Dependent Growth Adaptation Kinetics in 3T 3 Cell Populations Following Sudden [Ca2+] and Temperature Changes. A Comparison with SV40-3T3 Cells

Lowered extracellular [Ca2+] cause low growth rates and low stationary cell densities in 3T3 cell cultures as compared to physiological [Ca2%]. Under otherwise constant conditions the extracellular [Ca2+] determines a stable stationary cell density, which can be reached by increase of net cell number or decrease of net cell number, depending on cell density at the time of [Ca2+] adjustment. SV40-3T3 cells do not show this [Ca2+] dependency. At 39 degrees C 3T3 and SV40-3T3 cell populations show an increased growth rate at low cell densities as compared to cell populations at 35 degrees C. Approaching the stationary density the growth rate of both cell sorts is reduced faster at 39 degrees C than at 35 degrees C, leading to lower stationary cell densities at 39 degrees C than at 35 degrees C. A temperature change from 39 degrees C to 35 degrees C or in the opposite direction can affect the stationary cell density of 3T3 cell populations only if applied before reduction of growth rate by density-dependent growth-inhibiting principles has taken place.

Decrease of saturation density of cells of hamster cell lines after treatment with dextran sulfate

Dextran sulfates of various molecular weights were added to cultures of 3 transformed cell lines of hamster, 3T6 cells and embryonic fibroblastic cells. Dextran sulfate of high molecular weight reduced the saturation densities of all the cell lines of hamster and 3T6 cells, but those of low molecular weight did not. The mitotic rate of the treated cells decreased at stationary cell density. Dextran sulfate had no effect on the growth of normal fibroblastic cells derived from mouse and hamster embryos. Viability of treated cells was indicated by the following results. Cells of cultures seeded at different cell densities grew at almost the same rate in the presence of dextran sulfate. Treated cells remaining in the monolayer stage began to grow after removal of dextran sulfate. The colony formation rate of treated cells was the same as that of untreated cells. With the exception of one cell line, the morphology of cells treated with dextran sulfate of high molecular weight was more flattened and there was less overlapping than in untreated cells. Treated cells were less agglutinable to concanavalin A than were untreated cells. These results suggest that dextran sulfate affects the cell surface, resulting in the decrease of saturation density of cell lines.