Synchronous cultures from the baby machine. A model for animal cells

A baby-machine system that produces newborn Escherichia coli cells from cultures immobilised on a membrane was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth, and a model designed to characterise the nature and quality of the synchrony of such cells in a quantitative manner has been published. The baby machine has now been adapted for animal cells, and the present article is an attempt to modify the model to include these cells as well. The model consists of five elements, giving rise to five adjustable parameters (and a proportionality constant): a major, essentially synchronous group of cells with ages distributed normally about zero; a minor, random component from a steady-state population on the membrane that had undergone only very little age selection during the elution process; a fixed background count, to allow for the signals recorded by the electronic particle counter produced by debris and electronic noise; a time-shift, to account for differences between time of cell division and end of sample collection; and the coefficient of variation of the interdivision-time distribution, taken to be reciprocal-normal. It is this last feature, a reciprocal-normal rather than a Pearson type III interdivision-time distribution, that distinguishes this version of the model from its predecessor. The model is fitted by unconstrained non-linear least-squares to data from three different leukemia cell lines. The standard errors of the parameters are quite small in all cases, making their estimates highly significant; the quality of the fit is striking. The five parameters of the model can be divided into two nuisance parameters, two that are associated with the methodology and one that describes an inherent property of the cell itself; it turns out that both methodology parameters are zero in all three data sets studied. We also discuss the partition of the transition-time dispersion between the age distribution of the newborn cells and the age distribution of dividing cells and show that a reliable estimate of the corresponding parameters requires an experiment that extends over at least two and a half doubling times.     

Cell-cycle research with synchronous cultures: an evaluation

The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth. In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates. Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony. We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state. The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.     

The effect of temperature on membrane hydraulic conductivity

The objective of this study was to use the temperature dependence of water permeability to suggest the physical mechanisms of water transport across membranes of osmotically slowly responding cells and to demonstrate that insight into water transport mechanisms in these cells may be gained from easily performed experiments using an electronic particle counter. Osmotic responses of V-79W Chinese hamster fibroblast cells were measured in hypertonic solutions at various temperatures and the membrane hydraulic conductivity was determined. The results were fit with the general Arrhenius equation with two free parameters, and also fit with two specific membrane models each having only one free parameter. Data from the literature including that for human bone marrow stem cells, hamster pancreatic islets, and bovine articular cartilage chondrocytes were also examined. The results indicated that the membrane models could be used in conjunction with measured permeability data at different temperatures to investigate the method of water movement across various cell membranes. This approach for slower responding cells challenges the current concept that the presence of aqueous pores is always accompanied by an osmotic water permeability value, P(f)>0.01 cm/s. The possibility of water transport through aqueous pores in lower-permeability cells is proposed.     

Estimation of the disruption in freeze-pressed Saccharomyces cerevisiae by an electronic particle counter

An electronic particle counter has been used to estimate the disintegration by freeze-pressing baker's yeast. A counter threshold level which just yielded the maximum count for intact cells was selected. The conductivity of the suspending medium was chosen such that maximum counts were obtained. Under these conditions, the electronic counts agreed well with the visual counts. At a certain threshold level the maximum count was obtained at a lower resistivity (higher conductivity) in the suspending solution with the freeze-pressed suspension than with untreated cells, indicating that damage to the permeability barrier may occur without disruntion of the cell envelope. Fresh baker's yeast cells do not behave as nonconducting particles. This has to be taken into account when volume determinations with electronic particle counters are performed.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Permeability of human granulocytes to dimethyl sulfoxide

The permeability of the membrane of human granulocytes to the permeating solute dimethyl sulfoxide (DMSO) was studied using the Onsager form of the phenomenological equations derived from the theory of irreversible thermodynamics. Changes in cellular volume were monitored with an electronic particle counter as samples of that population were introduced into hypertonic osmotica. Temperature and concentration sensitivity analyses of the permeability coefficients were carried out. It is shown that the introduction of the Onsager formalism allows further insight into the observed transport phenomena. It was found that DMSO may affect the water permeability properties of the membrane for that population of cells.     

Transport,metabolism and distribution space of octanoate in the perfused rat liver

The scope of the present work was to investigate the metabolism and the passage of octanoate from albumin into the phospholipid bilayer of the plasma membrane and from thence into the cell space. The experiments were done in the isolated perfused rat liver with infusions of albumin and octanoate at various concentrations. Once steady-state conditions were attained, trace amounts of [1-¹⁴C]-octanoate, [¹³¹I]-albumin and [³H]-water were injected simultaneously and the effluent perfusate was fractionated. The normalized dilution curves were used for model analysis. The model which gives the best fit to the experimental results and which also produces the most consistent parameters is one that presupposes a rapid distribution of octanoate into the cell membrane and a slow transfer from the cell membrane into the cytosol. The concentration dependence of the distribution between the membrane and the extracellular space is parabolic, suggesting that octanoate changes the properties of the cell membrane when present at higher concentrations. The passage from the cell membrane into the cell space is relatively slow and limits metabolic transformation partly or totally, depending on the octanoate concentration in the plasma membrane. The rapid transfer of octanoate from the albumin space into the plasma membrane corroborates previous measurements of the dissociation of the albumin–octanoate complex. © 1997 John Wiley & Sons, Ltd.     

A microquantitative method to characterize lectin-induced cytoagglutination

A microquantitative method to characterize lectin-induced cytoagglutination reactions is described. The assay, which utilizes an electronic particle counter, measures the disappearance of single cells from cell suspensions containing various concentrations of lectin. The method is sensitive, requiring only μg quantities of lectin, reproducible, and amenable to thorough statistical analysis. It was also utilized to quantitate the inhibition of lectin-induced cytoagglutination by various saccharides. The method was employed to characterize ConA-induced agglutination of guinea pig erythrocytes and Novikoff ascites hepatoma cells, and to investigate several parameters which influence lectin-induced cytoagglutination reactions.     

Improved analysis of hemagglutination assays for quantitation of lectin activity

A method to quantitate lectin activity based on hemagglutination assay in microtiter plates is described. In addition to the normal method of visual titer evaluation an electronic particle counter is used for counting of nonagglutinated cells in the microtiter wells; this allows a rapid, quantitative determination of the amount of lectin required to agglutinate 50% of the countable single cells. It is also recommended that counting results should be related to a standard curve of concanavalin A to improve the reproducibility of the assay.     

Effect of temperature on the size of Escherichia coli cells.

The distributions of cell volumes of steady-state Escherichia coli ML30G cultures at various temperatures were measured. For cultures in a minimal medium, the distributions were indistinguishable at several temperatures between 15 and 30 C; at higher temperatures the cells were slightly smaller, and at lower temperatures they were slightly larger. For cultures in a complex medium, the cells were slightly larger at both high and low temperatures of growth. An abrupt change of temperature within the middle range led to a transient change in the distribution of cell volume, suggesting that the size of dividing cells is well regulated. No synchrony of division was induced by a change in temperature.     

Cell kinetics in the erythroid compartment of guinea pig bone marrow: a model based on 3H-TdR studies

A model of steady-state erythropoiesis in the guinea pig is described. The model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic, polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labeling curves obtained in pulse and intermittent labeling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. The results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. The fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.     

Modeling the Carotid Sinus Baroreceptor

A mathematical model that describes the relationship between sinus pressure and nerve discharge frequency of the carotid sinus baroreceptor is presented. It is partly based upon the single-fiber data obtained by Clarke from the sinus nerve of a dog. The model takes into account what is currently known about the physiology of the baroreceptor. It consists of two nonlinear ordinary differential equations and eight free parameters. With one set of values for these eight parameters, the model reproduces well the experimental results reported by Clarke for positive ramp pressure inputs. Only three parameters needed to be adjusted in order to fit the dynamic data. The remaining five were obtained from static and steady-state data.     

Estimation of intracellular fluid volume of L cells

Dilution of ¹⁴C-sucrose solution by intracellular fluid released as a result of ultracentrifugation was used to estimate the intracellular fluid volume of L cells. Consistent relationships to total cell volume as estimated by use of an electronic particle counter were obtained. Expressed as a percentage of total cell volume, the mean value plus or minus the S.D. for 6 experiments was 72.8 ± 0.9.     

Methods for the determination of adipose cell size in man and animals

Four methods for the sizing of adipose cells in small samples of human or animal adipose tissue are compared. These methods depend on the preparation of cell suspensions by incubation of the tissue with collagenase or by prolonged fixation with osmium tetroxide and separation of the fixed cells. A Coulter electronic counter was used to count and size the suspended cells and a Zeiss particle size analyzer for the sizing of cells in photomicrographs. The use of the Coulter counter to count cells in a suspension derived from a known amount of tissue and subjected to osmium tetroxide fixation is recommended for accuracy and general applicability to adipose cells of all sizes in man and animals.     

Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.     

A model of the sodium dependence of dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in rat striatal synaptosomes as a function of both [DA] and [Na]. Carrier mediated uptake is totally dependent on external sodium. The data were fitted to a rapid equilibrium model which has been found in previous studies to fit, with appropriate simplification, uptake data for glutamate, GABA, and choline in several brain regions under varying conditions. This model also gives a good fit to the dopamine data. The minimal best fit simplification of this model allows for DA uptake along with two sodium ions and predicts that apparent maximal velocity of uptake should increase with [Na], while the Michaelis-Menten constant should decrease. The minimal best fit model for DA, and a number of kinetic parameters which quantitate the model, are compared to those for the GABA, glutamate, and choline transporters. The results are consistent with a symmetrical, rapid equilibrium model, which has been presented previously for other neurotransmitters and precursors (18). This model offers a unifying basis for understanding the sodium and membrane potential dependence of neurotransmitter transport and the possible participation of transporters in depolarization induced release throughout the CNS.     

On the Correlation of the Volume of a Prolate-Spheroidal Cell, Tetrahymena as Determined by Electronic Particle Counters and by Morphometry

ABSTRACT. The cell volume provided by electronic particle counters may be incorrect. As a particle, or cell, passes the counting device, its volume is calculated as a sphere. The electronically derived, mean cell volume (electronic MCV) of a population of Tetrahymena (prolate spheroid) is smaller than the volume (morphometric MCV) calculated from measured cell length and width. This discrepancy was studied using a Coulter Multisizer particle counter and cell morphometry. The electronic MCV averaged 0.70 of the morphometric MCV (1.00) but changed from 0.72 (fast growth) to 0.63 and 0.76 (slow or no growth) for cells having a mean length/width of 2.05, 2.33, and 1.61, respectively. The measured diameter of latex particles (used for calibration) was identical to that stated, but the diameter of the electronic MCV was larger than the width of the cells which related to wehther the length/width of the cells was above, or below, 2.00. Hence, electron particle counters register primarily the width of a prolate-spheroidal cell, oriented with its long axis in the direction of flow, and uses this value as diameter for the calculated sphere, whereas for more spherical cells, tumbling without any orientation, a mean of the axes is used. Factors for correction of the electronic MCV of Tetrahymena are provided.     

A fit to nerve membrane rectification curves with a double-dipole-layer membrane model

Following Wei's suggestion that nerve stimulation and conduction properties are due to dipole layers at the two membrane surfaces (Wei, 1969), we have done steady-state electro-diffusion calculations in the constant field approximation for a simple double-dipole-layer model. We are thereby able to quantitatively fit the recent potassium iso-osmotic rectification curves of Gilbert and Ehrenstein for the squid giant axon membrane. For the squid axon membrane in a natural ion environment, only the outside dipole layer is present in the fit to the data.     

Failure of Delayed Feedback Deep Brain Stimulation for Intermittent Pathological Synchronization in Parkinson’s Disease

Suppression of excessively synchronous beta-band oscillatory activity in the brain is believed to suppress hypokinetic motor symptoms of Parkinson’s disease. Recently, a lot of interest has been devoted to desynchronizing delayed feedback deep brain stimulation (DBS). This type of synchrony control was shown to destabilize the synchronized state in networks of simple model oscillators as well as in networks of coupled model neurons. However, the dynamics of the neural activity in Parkinson’s disease exhibits complex intermittent synchronous patterns, far from the idealized synchronous dynamics used to study the delayed feedback stimulation. This study explores the action of delayed feedback stimulation on partially synchronized oscillatory dynamics, similar to what one observes experimentally in parkinsonian patients. We employ a computational model of the basal ganglia networks which reproduces experimentally observed fine temporal structure of the synchronous dynamics. When the parameters of our model are such that the synchrony is unphysiologically strong, the feedback exerts a desynchronizing action. However, when the network is tuned to reproduce the highly variable temporal patterns observed experimentally, the same kind of delayed feedback may actually increase the synchrony. As network parameters are changed from the range which produces complete synchrony to those favoring less synchronous dynamics, desynchronizing delayed feedback may gradually turn into synchronizing stimulation. This suggests that delayed feedback DBS in Parkinson’s disease may boost rather than suppress synchronization and is unlikely to be clinically successful. The study also indicates that delayed feedback stimulation may not necessarily exhibit a desynchronization effect when acting on a physiologically realistic partially synchronous dynamics, and provides an example of how to estimate the stimulation effect.     

Cell Kinetics In the Erythroid Compartment of Guinea Pig Bone Marrow: A Model Based On 3H-Tdr Studies

A model of steady-state erythropoiesis in the guinea pig is described. the model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic, polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labeling curves obtained in pulse and intermittent labeling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. the results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. the fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.