A population balance model describing the cell cycle dynamics of myeloma cell cultivation

A multi-staged population balance model is proposed to describe the cell cycle dynamics of myeloma cell cultivation. In this model, the cell cycle is divided into three stages, i.e., G1, S, and G2M phases. Both DNA content and cell volume are used to differentiate each cell from other cells of the population. The probabilities of transition from G1 to S and division of G2M are assumed to be dependent on cell volume, and transition probability from S to G2M is determined by DNA content. The model can be used to simulate the dynamics of DNA content and cell volume distributions, phase fractions, and substrate and byproduct concentrations, as well as cell densities. Measurements from myeloma cell cultivations, especially the FACS data with respect to DNA distribution and cell fractions in different stages, are employed for model validation.     

Fetal erythroid cell development: density gradients and size distributions

Electronic size distributions of erythroid cells from fetal C57BL-6J mice during the eleventh through twentieth days of gestation indicate that the erythropoietic cell populations are constantly changing. The mean volume of the liver derived non-nucleated erythroid population decreases from four times the mean adult erythrocyte volume on the thirteenth gestation day to twice the adult erythrocyte volume at birth. The mean volume of the nucleated erythroid cell is about ten times the mean adult erythrocyte volume. The gestation age of an embryo can be determined from blood cell size distributions. The mode of the non-nucleated population and the percentage of each population indicates the gestation age. Size distribution of cells in density gradient fractions apparently indicate two size populations of non-nucleated cells between the thirteenth and fifteenth days. The density of the non-nucleated cells increases during gestation. It is suggested that the decrease in size and increase in density of non-nucleated cells is due to the release of successively smaller reticulocytes from the liver.     

Kinetic characterization of baculovirus-induced cell death in insect cell cultures

The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (t(d)) and a first-order death phase characterized by a half-life (t(1/2)). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection (c) 1993 John Wiley & Sons, Inc.     

Fluorimetric multiparameter cell assay at the single cell level fabricated by optical tweezers

A fluorimetric multi-parameter cell sensor at the single cell level is presented which makes it possible to observe the physiological behavior of different cell lines, different physiological parameters, and statistical data at the same time. Different cell types were immobilized at predefined positions with high accuracy using optical tweezers and adhesion promoting surface layers. The process is applicable to both adherent and non-adherent cells. Coating of the immobilization area with mussel adhesive protein was shown to be essential for the process. Intracellular proton and calcium concentrations in different cell classes were simultaneously imaged and the specific activation of T lymphocytes was demonstrated. This method should be especially useful for drug screening due to the small sample volume and high information density.     

Growth of Streptococcus faecalus in dense culture

A fermentation system was designed and constructed to study the growth characteristics of microorganisms at low and high cell concentrations. The technique used to develop high cell densities utilized a rotating microfiltration unit to permit the removal of cell-free product from the fermenter. The fermenter volume and the filter were contained in a single unit composed of a series of concentric cylinders. Annuli contained the fermenter volume while the second outermost cylinder supported a microfiltration membrane. Feed to the system was pumped at constant rates, and the internal pressure built up to a value, which would effect the required filtration rate. The system was operated batchwise and continuously with and without filtration. The anaerobie growth characteristics of Streptococcus faccalus were determined at 37°C and pH 7.0 for batch, continuous, and continuous with filtration modes of operation. The growth characteristics were unchanged when the cell density was increased. Changes in cell yield peer model of glucose consumed were clearly illustrated during thee course of single run by operating the fermenter in the unsteady state with filtration. No consumption of glucose for developed was 40% packed cell volume, a value 45 times larger than could be grown in simple batch culture.     

Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa.

Cells isolated from the rat gastric mucosa were resolved into two fractions on a percoll density gradient, and into five fractions using counterflow centrifugation (elutriation). Ca(2+)-dependent nitric oxide synthase (NOS) activity was found in the high density percoll fraction but not in the parietal cell enriched low density fraction. This activity was inhibited by NG-monomethyl-L-arginine with an IC50 of 3.7 microM. Cells in the elutriator fraction rich in mucous-epithelial cells exhibited the highest NOS activity, while the smaller cell fractions had no detectable NOS yet had the highest basal release of prostaglandin E2. The parietal cell enriched elutriator fraction again had low NOS activity. The activity of a constitutive NOS in the mucous-cell fraction may indicate a role of NO in the regulation of epithelial cell integrity or secretion.     

Increases in platelet and red cell counts,blood viscosity,and arterial pressure during mild surface cooling: factors in mortality from coronary and cerebral thrombosis in winter.

Six hours of mild surface cooling in moving air at 24 degrees C with little fall in core temperature (0.4 degree C) increased the packed cell volume by 7% and increased the platelet count and usually the mean platelet volume to produce a 15% increase in the fraction of plasma volume occupied by platelets. Little of these increases occurred in the first hour. Whole blood viscosity increased by 21%; plasma viscosity usually increased, and arterial pressure rose on average from 126/69 to 138/87 mm Hg. Plasma cholesterol concentration increased, in both high and low density lipoprotein fractions, but values of total lipoprotein and lipoprotein fractions were unchanged. The increases in platelets, red cells, and viscosity associated with normal thermoregulatory adjustments to mild surface cooling provide a probable explanation for rapid increases in coronary and cerebral thrombosis in cold weather. The raised arterial pressure and possibly cholesterol concentration may contribute to slower components of the increased thrombosis.     

Differential effects of interleukin 2 vs B cell growth factor on human B cells

The effects of recombinant interleukin 2 (IL-2) and high m.w. (HMW) B cell growth factor (BCGF) were examined on normal human peripheral blood B cells activated with Staphylococcus aureus Cowan I (SAC). When SAC-activated B cells were separated into Tac-antigen (Tac-Ag)+ and Tac-Ag- fractions by a cell sorter, recombinant IL-2 induced only the Tac-Ag+ cells to proliferate, whereas both Tac-Ag+ and Tac-Ag- cells responded to HMW-BCGF (m.w. 60,000). Alternatively, SAC-activated B cells were separated according to density into three fractions: low density (large) cells (82 +/- 15% Tac-Ag+), intermediate density (medium) cells (45 +/- 13% Tac-Ag+), and high density (small) cells (less than 5% Tac-Ag+). Recombinant IL-2 enhanced proliferation of low density cells the most, intermediate density cells less, and high density cells not at all. HMW-BCGF induced all three fractions to proliferate to approximately the same degree. Finally, the effects of IL-2 and BCGF on the DNA and RNA content of the various fractions of B cells was examined. RNA content was greater in IL-2-stimulated B cells than BCGF-stimulated B cells, whereas DNA content was the same in both cell populations. IL-2 and BCGF may preferentially interact with different subpopulations of B cells. The interaction of IL-2 or BCGF with normal activated B cells may induce both similar and different intracellular events.     

Condition-dependent cell volume and concentration of Escherichia coli to facilitate data conversion for systems biology modeling

Systems biology modeling typically requires quantitative experimental data such as intracellular concentrations or copy numbers per cell. In order to convert population-averaging omics measurement data to intracellular concentrations or cellular copy numbers, the total cell volume and number of cells in a sample need to be known. Unfortunately, even for the often studied model bacterium Escherichia coli this information is hardly available and furthermore, certain measures (e.g. cell volume) are also dependent on the growth condition. In this work, we have determined these basic data for E. coli cells when grown in 22 different conditions so that respective data conversions can be done correctly. First, we determine growth-rate dependent cell volumes. Second, we show that in a 1 ml E. coli sample at an optical density (600 nm) of 1 the total cell volume is around 3.6 μl for all conditions tested. Third, we demonstrate that the cell number in a sample can be determined on the basis of the sample's optical density and the cells' growth rate. The data presented will allow for conversion of E. coli measurement data normalized to optical density into volumetric cellular concentrations and copy numbers per cell--two important parameters for systems biology model development.     

Interrelations among Na and K content,cell volume,and buoyant density in human red blood cell populations

Summary This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na+K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966.Scand. J. Lab. Invest. 18:167–180) cell water and cell volume was found to vary directly with the summed content of Na+K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na+K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Human red blood cell aging: correlative changes in surface charge and cell properties

Red blood cells (RBCs) during microcirculation, aging and storage, lose N-acetylneuraminic acid (NANA) and other biomaterials thereby altering cell structures, some properties and functions. Such cell damage very likely underlies the serious adverse effects of blood transfusion. However, a controversy has remained since 1961-1977 as to whether with aging, the RBCs, suffering loss of NANA, do have a decreased charge density. Any correlation between the changes in the cell properties with cell aging is also not clear. Therefore, to remove the ambiguity and uncertainty, we carried out multiparameteric studies on Percoll fractions of blood of 38 volunteers (lightest-young-Y-RBCs, densest-old-O-RBCs, two middle fractions).We found that there were striking differences between the properties of Y-RBCs and O-RBCs. The ζ-potential of Y-RBCs decreased gradually with aging. Studies in parallel on RBC fractions incubated with both positively charged quantum dots and Sambucus Nigra-fluorescein isothiocyanate (FITC) along with their ζ-potentials provide for the first time direct visual evidence about the lesser amount of charge density and NANA on O-RBCs, and a collinear decrease in their respective ζ-potentials. Close correlation was found between the surface charge on an aging RBC and its structure and functions, from the cell morphology, the membrane deformability to the intracellular Hb structure and oxidation ability. This quantitative approach not only clarifies the picture but also has implications in biology and medicine.     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.     

Regulation of antigen-induced lymphoproliferation by BSA gradient-separated T cell fractions

Human T cells from 6 volunteers immunized with keyhole limpet hemocyanin (KLH) or tetanus toxoid were fractionated on bovine serum albumin (BSA) gradients. These T cell fractions were then recombined with autologous unseparated mononuclear cells or unseparated T cells to determine the effect of each fraction on the proliferative (DNA synthetic) response to the immunizing antigen. Using this separation technique, we were able to define 2 fractions of T cells of widely different densities that had a suppressive effect on the proliferative response to KLH of unfractionated T cells. The demonstration of suppressive activity was possible only if cells were tested within 8 wk of immunization with KLH. These suppressive T cell fractions differed not only in density but also in surface characteristics. Fraction 1 cells had a high proportion of Ia+ cells but no Fc receptors for IgG (FcRG-) were seen, whereas cells from fraction 4 (of higher density) were Ia- and were composed of 12.1 +/- 1.2% FcRG+ cells. this suppression was shown to be specific for the immunizing antigen. One individual who had been immunized with KLH for more than 1 yr in which suppression of KLH-induced proliferation was no longer demonstrable was given primary immunization to tetanus toxoid. Suppression could be demonstrated in fractions 1 and 4 specific for tetanus toxoid; there was no demonstrable effect of these T cell fractions on the response to KLH. We postulate that these are different functional populations of suppressor T cells that regulate antigen specific lymphoproliferation.     

Two-dimensional impedance studies of BSA buoyant density separated human erythrocytes

Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.     

Normally occurring inhibitory cells for natural killer cell activity. I. Organ distribution

Analysis of natural killer (NK) activity in different organs from mice or rats fractionated using discontinuous density gradients have revealed typical distinct density profiles according to the organ from which the NK cells were derived. High NK level organs thus tended to have significant lytic activity extending into 1.090 density fractions whereas the low NK population had its peak activity more strictly confined to the 1.067 fraction. The reason for this skewedness we find to be dependent upon the presence of an inhibitor cell for NK cells to be found in the higher-density fractions. The significance of this inhibitor cell was more apparent when NK activity was low, thus skewing peak levels to lower fractions. The activity of this inhibitory cell was not found to vary with age, thus failing to explain the age-dependent rise and fall of NK activity in rodents. Presence of such an inhibitory cell also explains why sizeable NK activity can be disclosed in a fraction of cells obtained from a population which before fractionation failed to disclose NK function.     

Influence of sulfur accumulation and composition of sulfur globule on cell volume and buoyant density of Chromatium vinosum

Average cell volume and cell buoyant density of Chromatium vinosum DSM 185 growing in sulfide limited continuous cultures, were found to increase with increasing dilution rate. It was found that the increase in buoyant density was mainly a consequence of the accumulation of elemental sulfur. The contribution of other compounds such as protein, bacteriochlorophyll a and glycogen, was almost negligible. It was concluded that the sulfur globule is constituted by at least two fractions, sulfur and an unidentified moiety with a density lower than that of sulfur, probably water.A model was developed to explain the relation between the specific content of sulfur and cell buoyant density. The model also predicts the impact of elemental sulfur on the volume of the cell. It was found that in addition to the accumulation of sulfur the average cell volume also changes with the specific growth rate.In shift-up experiments (sulfur accumulation) the actual phenomena agreed with those predicted by the model, however, this was not so during shift-down (sulfur depletion). It is suggested that this difference is due to the fact that during the shift-down, elemental sulfur and the unidentified moiety are being depleted at different rates.Non-standard abbreviations BChl bacteriochlorophyll - PHB poly--hydroxybutyric acid - D dilution rate - specific growth rate - S _R reservoir concentration of limiting substrate     

Interaction of rifampicin with hepatocyte organoids in rats and its intracellular distribution

Interaction of rifampicin with isolated subcellular fractions of the rat liver (binding and dissociation of the complexes) and intracellular distribution of the antibiotic were studied in the presence of the main organoids taken in the ratio close to the natural volume ratio of the hepatocyte cell components. The nuclei and mitochondria were most active during drug binding. The microsomes were less important. Rifampicin formed mobile complexes with organoids and was easily released from the subcellular fractions recovering its activity on washing. The intracellular distribution was the following: 37.7 per cent of rifampicin in the active form was accumulated in cytosol and the remaining amount was reversibly bound in the fractions of the nuclei, mitochondria and microsomes. The characteristic features of the cell pharmacokinetics of rifampicin, i.e. significant concentration in cytosol, possible deposition in the subcellular structures and at the same time the capacity for recovery of the activity might define the antimicrobial potential of this antibiotic in respect to the intracellular microorganisms.     

Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines

Data on viscous (eta') and elastic (eta') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 mum as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 mum mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, \documentclass{article}\pagestyle{empty}\begin{document}$ \eta = \eta _0 \exp [K\dot \gamma ;{ - \beta } \phi /(1 - K'\sigma \phi _c /D)] $\end{document} was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field. (c) 1993 John Wiley & Sons, Inc.