Growth In Continuous Culture Research Articles

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro.

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro.

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

Kinetic study of hybridoma cell growth in continuous culture: II. Behavior of producers and comparison to nonproducers

A hybridoma cell line, AFP-27-P, was cultivated in continuous culture under glucose-limited conditions. The viable cell concentration, dead-cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP-27-NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod-type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon-growth-associated term and a negative-growth-associated term. The values for the model parameters were determined from regression analysis of the steady state data.

A model for light‐limited continuous cultures: Growth, shading, and maintenance

Light-limited growth in continuous cultures of phototrophic organisms is modeled. It is assumed that light energy up-take rate depends hyperbolically on light intensity and that the maintenance costs are proportional to biomass. Modeling the light distribution caused by shading within the vessel is necessary to explain the existence of steady state in light-limited chemostats. The model fits well to experimental data from literature on light-limited chemostats and turbidostats. Attention is given to the implications of the model for the estimation of the specific maintenance rate constant in light-limited continuous cultures.

Selection of colony, plasmid, and virulence variants of Staphylococcus epidermidis NRC853 during growth in continuous cultures exposed to erythromycin

A continuous-culture system was developed to study changes in the structure of Staphylococcus epidermidis populations exposed to subminimum inhibitory concentrations of erythromycin. Continuous-culture experiments were carried out in a dextrose-free, tryptic soy broth medium supplemented with lactic acid and sodium lactate (MTSB-D). The multiresistant (penicillin-, tetracycline-, and erythromycin-resistant) S. epidermidis strain NRC853 was subjected to a series of experiments: (i) growth individually in continuous culture in the absence and presence of erythromycin and (ii) growth in mixed culture with the erythromycin-susceptible S. epidermidis strain NRC852 in the absence and presence of erythromycin. Strain NRC853 produced colony morphology variants during continuous culture in the presence of 0.05 and 0.1 microgram of erythromycin per ml. Variants (A, B, and C) were different from their wild-type parent on the basis of colony size, sector pattern, and/or the ability to transmit light. A variants rapidly lost a 2.7-MDa tetracycline resistance plasmid. B and C variants formed an ermC plasmid multimer series from unit size to a 16-mer and exhibited an approximately twofold increase in erythromycin MIC over that of the wild-type parent. They slowly lost the tetracycline resistance plasmid. The small-colony B variant demonstrated an increased virulence in the neonatal mouse weight gain test and an increase in fibronectin binding compared with the wild-type parent. The presence of a competing strain drastically increased the frequency of all variants.

Myointimal Hyperplasia: Pathogenesis and Implications. 1. In Vitro Characteristics

Myointimal hyperplasia (MIH) is an arterial wall smooth muscle cell (SMC) proliferative disorder. This process is responsible for a significant number of early and long-term arterial reconstructive and graft failures. Histopathologically, this process is characterized by a proliferation of SMC in the intima of traumatized arteries resulting in arterial and/or anastomatic stenosis with secondary thrombosis. In vitro studies of cultured SMC have allowed the evaluation of SMC response to factors suspected of being clinically associated with MIH. Principal among these is platelet derived growth factor (PDGF), which is known to be secreted by several cell types including endothelial cells (ECs) and monocytes as well as being stored and secreted by platelets. PDGF, somatomedin-C, epithelial growth factor, insulin, and other factors have been found to significantly increase SMC replication in vitro. Lipoproteins may be important substrates for SMC proliferation in contrast to heparin, which may directly inhibit SMC protein synthesis. Unlike SMCs, whose continued growth in culture is dependent on various growth factors and nutrients, ECs essentially cease to proliferate after the cells have formed a monolayer over the available surface. Extracellular matrix proteins, polypeptide mitogens, and heparin have been shown to modify EC migration and proliferation in vitro. Wounding of EC monolayers by scratching results in increased replication and migration, processes which require plasma factors that remain poorly defined. However, two general forms of EC growth factor have been isolated from many body tissues, are potent stimulators of capillary endothelial growth, and appear important both for normal EC monolayer homeostasis and for the response to injury. Cultured ECs produce mitogens for SMC. Production of the principal mitogen, PDGF, is significantly increased in sparse versus confluent cell cultures as well as by toxic agents such as endotoxin and phorbol esters. Acetyl low density lipoprotein as well as omega-3 fatty acids may significantly and selectively inhibit EC PDGF production, a finding with potentially profound implications for the clinical control of MIH in vivo.

Kinetic study of hybridoma cell growth in continuous culture. I. A model for non‐producing cells

The kinetic behavior of a nonproducing hybridoma clone AFP-27-NP was investigated in continuous culture under glucose-limited conditions. A total of more than 21, 000 h of cultures were operated at dilution rates ranging from 0.01 to 0.06 h(-1). The viable cell concentrations, dead cell concentrations, and cell volumes all varied with the dilution rate. A steady-state model was developed based on the biomass concentration and the glucose concentration. The specific growth rate as a function of glucose concentration is described by a model similar to the Monod model with a threshold glucose concentration and a minimum specific growth rate incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. A death rate is included in the model which is described by an inverted Monod-type function of glucose concentration. The yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper region of specific growth rates. No maintenance term for glucose consumption was needed; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepted the specific growth rate axis at a value close to the minimum growth rate. The values for the model parameters were determined from regression analysis of the steady-state data. The model predictions and experimental results fit very well.

On multiple-nutrient-limited growth of microorganisms, with special reference to dual limitation by carbon and nitrogen substrates

Simultaneous limitation of microbial growth by two or more nutrients is discussed for dual carbon/nitrogen-limited growth in continuous culture. The boundaries of the zone where double-limited growth occurs can be clearly defined from both cultivation data and cellular composition and they can be also predicted from growth yield data measured under single-substrate-limited conditions. It is demonstrated that for the two nutrients carbon and nitrogen the zone of double nutrient limitation is dependent on both the C:N ratio of the growth medium and the growth (dilution) rate. The concept on double-(carbon/nitrogen)-limited growth presented here can be extended to other binary and multiple combinations of nutrients.

Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any given dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.

Maintenance of proton motive force by Streptococcus mutans and Streptococcus sobrinus during growth in continuous culture

The components of the transmembrane electrochemical proton gradient, or proton motive force (PMF, delta p), were determined in cells of Streptococcus mutans Ingbritt and Streptococcus sobrinus ATCC 27352 growing in continuous culture under conditions of changing glucose concentration, growth rate and growth pH. The pH gradient (delta pH) and membrane electrical potential (delta psi) were assayed with the weak acid, salicyclic acid, and the lipophilic cation, methyltriphenylphosphonium iodide, respectively. S. mutans Ingbritt growing in continuous culture (pH 7.0, dilution rate (D) = 0.1 h-1) at 8 glucose concentrations ranging from 2.8 to 288 mM maintained a relatively constant delta p of 58.3 mV (SD +/- 5.8) in spite of a transition from glucose to nitrogen-limited growth and significant changes in cell physiology. Changes included a decreasing yield constant, increasing glucose uptake rates in the chemostat, repression of Ellglc of the PEP phosphotransferase sugar transport system and decreasing glycolytic capacity of the cells as the medium glucose concentration increased. Changes in the dilution or growth rate of S. mutans Ingbritt from 0.1 to 1.0 h-1 and S. sobrinus from 0.1 to 0.8 h-1, when growing at pH 7.0 with limited glucose and lactose, respectively, resulted in significantly lower delta p values due to the dissipation of the delta psi. When the cells of S. mutans Ingbritt were grown with excess glucose (nitrogen limitation), lower delta p values were observed at pH 5.5, but not at pH 7.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of the LDL receptor in Epstein-Barr virus transformed lymphocytes

Non-dividing human lymphocytes were transformed upon infection with the Epstein-Barr virus (EBV) into lymphoblasts which are capable of continuous growth in culture. We studied the properties of the LDL receptor in EBV-transformed human lymphocytes (EBV-L) by binding experiments and by ligand blotting. EBV-L show a high affinity binding of LDL in the same order of magnitude as found with fibroblasts; EBV-L obtained from a homozygous familial hypercholesterolemic (FH) patient fail to express LDL receptor activity. Similar to that of fibroblasts, the LDL receptor activity in EBV-L is Ca 2+-dependent and is down-regulated by the presence of an exogenous source of cholesterol in the medium. The LDL receptor protein of EBV-L has an apparent molecular weight of 130000. Since our results show that EBV-L display a LDL receptor protein similar to the LDL receptor present in fibroblasts, we conclude that in comparison with other cell types the EBV-L offer a suitable model system to investigate LDL receptor protein abnormalities in FH patients.

Behavior of the hybrid plasmid pNSW301 in Zymomonas mobilis grown in continuous culture

The stability of the plasmid pNSW301 which was formed by cointegration of the Inc W R plasmid Sa and the 14.5-kb pNSW1 plasmid of Zymomonas mobilis ZM6100 was investigated in ZM6100(pNSW301) grown in continuous culture without antibiotic selection. The cointegrate plasmid, pNSW301, was found to be structurally unstable and a total reduction in the size of pNSW301 of approximately 21 kb occurred during growth in continuous culture. Following a systematic study, a number of deletion derivatives of pNSW301 were isolated and used to transform Escherichia coli HB101, with the exception of pNSW312. The plasmid pNSW312 was 100% stable in ZM6100(pNSW312) in continuous culture but was unable to replicate in E. coli.

A general model for aerobic yeast growth: Continuous culture

A general model for the aerobic growth of yeast in continuous culture is presented. The model is capable of simulating the complete range of metabolic responses observed for yeast growth in continuous culture including respiratory repression, saturated respiratory capacity, and respiratory depression.It is postulated that respiratory depression is the result of the adaptation (increase in capacity of the respiratory intermediate transport proteins located at the mitochondrial membrane). Respiratory repression and subsequent saturated respiratory capacity is postulated to be the result of the gradual transfer of biosynthetic intermediates provision from the mitochondrion to the cytoplasm or, possibly, the adaptation (increase) in the capacity of the cell to excrete ethnol. It is difficult to provide a definitive experimental verification of these postulates.Irrespective of the biochemical basis of respiratory repression and depression, the model described is capable of simulating the complete range of metabolic responses obtained for yeast growth in continuous culture. It is the only model reported in the literature capable of achieving this.

A mechanistic growth model of a methylotroph

AbstractBased on the biochemical knowledge of the RMP‐cycle methylotrophs and the experimental results of batch and continuous culture growth of a methylotroph, L3, a simple mechanistic growth model of L3 was developed. The growth model consists of a basic structure and a regulatory structure. The basic‐model structure was developed by incorporating formaldehyde as a state variable and including a lpump‐and‐leak' mechanism of formaldehyde transport. The regulatory‐model structure was built by acknowledging growth inhibition due to formaldehyde and formaldehyde inhibition of methanol oxidation. The resulting model describes adequately the batch as well as the stable and unstable steady‐state continuous culture behavior of L3 on mixtures of methanol and formaldehyde.

Model for the feedback control system of bacterial growth II. Growth in continuous culture

A mathematical model is developed that describes substrate limited bacterial growth in a continuous culture and that is based upon the conceptual framework elaborated in a previous paper for describing the feedback control system of cell growth [S. Bleecken, (1988). J. theor. Biol. 133, 37.] Central to the theory are the ideas that the limiting substrate is converted into low molecular weight building blocks of macromolecular synthesis which again are converted into biomass (RNA and protein) and that the rates of RNA and protein synthesis are controlled by the intracellular concentration of building blocks. It is shown that a continuous culture can be simulated by two interconnected feedback control systems the actuating signals of which are limiting substrate concentration and the intracellular concentration of building blocks, respectively. Three types of steady-states are found to appear in a continuous culture, besides the well-known stable steady-state of the whole culture there exist two batchlike steady-states of the biotic part of the culture which are metastable. The model is used to analyse the steady-states and their stability properties as well as the dynamic responses of biomass, RNA, protein, building block and substrate concentrations to changes in environmental conditions. Especially the inoculation of a continuous culture and the effects of step changes in dilution rate, inlet substrate concentration and growth temperature are studied in detail. Relations between the growth behaviour of a single cell and that of a continuous culture are derived. The RNA to protein ratio is introduced as a rough measure of the physiological state of cells and it is shown that a cell reacts to environmental changes with a simple pattern of basic responses in growth rate and physiological state. There are reasons to assume that the model presented is the minimal version of a structured model of bacterial growth and represents an optimum compromise between biological relevance and mathematical practicability.

Dilution Rates on the Efficiency of Rumen Microbial Growth in Continuous Culture

Dilution Rates on the Efficiency of Rumen Microbial Growth in Continuous Culture

The Production of Alginate by Pseudomonas mendocina in Batch and Continuous Culture

The effect of growth conditions on the production of alginate by highly mucoid strains of Pseudomonas mendocina was investigated, using pH-controlled batch and continuous cultures. In batch culture, alginate was produced both during the exponential and stationary phases of growth. A polysaccharide concentration of 20 g 1−1 was obtained from medium containing glucose (50 g l−1). In steady-state continuous culture under nitrogen limitation (dilution rate, D = 0·05 h−1), a similar alginate concentration was attained; more than 60% of the utilized glucose was converted to alginate and the polysaccharide concentration was ten-fold the biomass concentration. Increasing the dilution rate above 0·1 h−1 decreased the proportion of glucose converted to alginate although the biomass was unchanged. The optimum dissolved oxygen tension (d.o.t.) for alginate production in nitrogen-limited continuous culture was approximately 40 mm Hg (1 mm Hg is approximately 133 Pa). The respiration rate and bacterial density were independent of d.o.t. above 10 mm Hg. Under oxygen limitation, no alginate was produced but acetate was excreted into the culture fluid. Variants producing little alginate arose during growth in continuous culture. Under carbon limitation, alginate production was substantially decreased but not eliminated.

Capture and characterization of 5-aza-2'-deoxycytidine-treated C3H/10T1/2 cells prior to transformation.

A model system was developed to capture phenotypically normal cells committed to transformation to address two fundamental questions in cancer biology: (i) what are the earliest events in transformation; and (ii) what is the role of DNA methylation in carcinogenesis? Individual C3H/10T1/2 cells were treated with 5-aza-2'-deoxycytidine, which causes hypomethylation of DNA. Cells were grown to subconfluence, and individual microcolonies were trypsinized into two fractions. One fraction was cryopreserved, and the other was replated and maintained in culture. Ten percent of these replated colonies became morphologically transformed after 4-6 weeks. The cryopreserved ancestral cells of both transformed and nontransformed microcolonies were then cultured and compared to each other and to the transformants for phenotypic properties of cellular transformation. Pretransformed and nonpretransformed ancestral cells were initially morphologically indistinguishable, their early growth curves did not differ significantly, and they did not form colonies in soft agar. On continued growth in culture, however, the pretransformants displayed all of the phenotypic characteristics of transformation. Furthermore, transformation occurred in a given pretransformant in most or all of the cells of that clone. Thus, cells committed to transformation could be isolated in this way prior to phenotypic transformation. Studies of these pretransformed cells will permit examination of the earliest events in carcinogenesis and the role of DNA methylation in transformation.

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.

Inorganic nitrogen utilization by assemblages of marine bacteria in sea-water culture

Stimulation of heterotrophic bacterial growth by inorganic nitrogen (nitrate and ammonium) was observed in natural assemblages of marine bacteria growth in continuous culture with unsupplemented sea water as primary medium. In the presence of nitrogenous supplements, bacterial numbers increased approximately 3-fold. These results indicate that re-evaluation of the role of heterotrophic bacterioplankton in the pelagic nitrogen cycle may be necessary.