Normal human endometrium in cell culture. II. A microspectrophotometric study of polyploid nuclei in short-term primary epithelial cultures.

The growth of short-term primary cultures of endometrial epithelium has been studied using Feulgen microspectrophotometry. A gradual increase in the number of polyploid nuclei up to 64C has been observed and is associated with a decline in the growth capacity of the cultures. The specific mechanism(s) of this polyploidization is not known.     

A Method for Studying Photosynthetic Capacities of Unicellular Algae Based on in vivo Chlorophyll Fluorescence

The correlation between photosynthesis and DCMU-induced fluorescence increase has been studied in four species of unicellular green algae from inoculation of the cultures to the stationary growth phase. The fluorescence increase induced by DCMU was high in exponentially growing cultures but decreased when the cultures approached the stationary phase of growth. Because of the good correlation between photosynthesis and DCMU-induced fluorescence increase, the fluorescence technique is a promising method for describing changes in photosynthetic capacities of algal populations in natural waters.     

The energetics of bacterial growth: a reassessment

The growth yield of microbial cultures can be used to estimate the efficiency of energy generation during a fermentation or respiration, in the past, the assessment of this efficiency in organisms carrying out a respiration has been the subject of many heated debates. This has partly been caused by the complexity of microbial respiratory chains. Strains of Escherichia coli specifically modified in their respiratory chain have been used recently to re-evaluate the energetic efficiency of the bacterial respiration using chemostat cultures. The different strains indeed show different growth efficiencies. The physiological significance of energetically less-efficient branches of the respiratory chain is discussed.     

Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin

Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions, a common phenomenon in large-scale cultivations of bacteria. This technology has now been applied to tobacco suspension cultures. Tobacco suspension cultures have been generated from VHb-expressing tobacco plants. Cell cultures were capable of producing an active hemoglobin. When grown in shake flasks, the cells did not show any lag-phase and exhibited improved cell growth, compared to controls carrying the parental plasmid.     

Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth – associated production of a foreign protein, β-galactosidase

Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth of MDA-MB-231 cell line: different effects of TGF-beta(1), EGF and estradiol depending on the length of exposure.

The human cell line MDA-MB-231 is a prototype for the study of hormone-independent breast cancer. Modification of cell growth behaviour has been observed after treating these cells with growth factors. EGF is a typical stimulatory growth factor for many cell types, whereas transforming growth factor beta(1)(TGF-beta(1)) acts with inhibitory character. Here we observed cell growth inhibition after EGF as well as after TGF-beta(1)treatments. Nevertheless, in the 42-h experiments, EGF-treated cultures grew before (18 hours) respect to the TGF-beta(1)and E(2)-treated cultures (24 h), and in the 11-day experiments, EGF-treated cultures started growing (7 days) after TGF-beta(1)-treated cultures (5 days). Estradiol inhibited the proliferation of these cells only after several days of treatment.     

A method for turbidimetric measurement of bacterial growth in liquid cultures and agar plug diffusion cultures,using standard test tubes

Summary A method has been developed for turbidimetric measurement of bacterial growth in standard inexpensive test tubes with closures in-place. Liquid cultures and agar plug diffusion cultures can be assayed using an unmodified spectrophotometer. Growth curves of replicate cultures grown in test tubes, are reproducible with respect to similarity of curve shape, onset of logarithmic growth phase, and maximum growth.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

A model for noninhibitory microbial growth

A model for noninhibitory microbial growth has been developed which is superior to the Monod model in that it can predict the decline in steady-state growth yields at both the slow and the fast specific growth rates. The model parameters are evaluated from data obtained for steady-state, phenol-limited Pseudomonas putida growth using a conventional 1-dm(3) cheniostat. The model also has been successfully applied to Mor and Fiechter's data for cheniostat yeast cultures.     

Toxic effects of chromium(VI) on anaerobic and aerobic growth of Shewanella oneidensis MR-1

Cr(VI) was added to early- and mid-log-phase Shewanella oneidensis (S. oneidensis) MR-1 cultures to study the physiological state-dependent toxicity of Cr(VI). Cr(VI) reduction and culture growth were measured during and after Cr(VI) reduction. Inhibition of growth was observed when Cr(VI) was added to cultures of MR-1 growing aerobically or anaerobically with fumarate as the terminal electron acceptor. Under anaerobic conditions, there was immediate cessation of growth upon addition of Cr(VI) in early- and mid-log-phase cultures. However, once Cr(VI) was reduced below detection limits (0.002 mM), the cultures resumed growth with normal cell yield values observed. In contrast to anaerobic MR-1 cultures, addition of Cr(VI) to aerobically growing cultures resulted in a gradual decrease of the growth rate. In addition, under aerobic conditions, lower cell yields were also observed with Cr(VI)-treated cultures when compared to cultures that were not exposed to Cr(VI). Differences in response to Cr(VI) between aerobically and anaerobically growing cultures indicate that Cr(VI) toxicity in MR-1 is dependent on the physiological growth condition of the culture. Cr(VI) reduction has been previously studied in Shewanella spp., and it has been proposed that Shewanella spp. may be used in Cr(VI) bioremediation systems. Studies of Shewanella spp. provide valuable information on the microbial physiology of dissimilatory metal reducing bacteria; however, our study indicates that S. oneidensis MR-1 is highly susceptible to growth inhibition by Cr(VI) toxicity, even at low concentrations [0.015 mM Cr(VI)].     

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures ("confluent-log" cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that "confluent-log" cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.     

Yield depression in methanol-utilizing batch cultures

Summary Yield depression, as opposed to growth inhibition, in batch cultures of methanol-utilizing microorganisms is discussed. Under conditions where the yield coefficient varies, the effect on oxygen demand has been predicted for exponentionally growing cultures.     

The growth of a cyanide-utilising strain of Pseudomonas fluorescens in liquid culture on nickel cyanide as a source of nitrogen

Pseudomonas fluorescens strain NCIMB11764 is able to utilise cyanide as a source of nitrogen for growth. When KCN(≡ HCN) is the source of nitrogen it has to be supplied as the limiting nutrient in fed-batch cultures [1]. In this study it has been shown that metal-complexed cyanide, as nickel cyanide (Ni(CN)²⁻₄), can be used as the source of nitrogen when it is added directly to the growth medium in batch cultures. Ni(CN)²⁻₄ could also be used as the source of nitrogen in nitrogen-limited continuous cultures. In both batch and continuous cultures, growth on Ni(CN)²⁻₄ was associated with induction of cyanide oxygenase activity. An assay for cyanide has been developed utilising its binding to nickel.     

Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures

As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G(1) phase of the cell cycle. The steady-state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady-state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. (c) 1992 John Wiley & Sons, Inc.     

Growth induction by serum or polyoma virus inhibits the aggregation of trypsinised suspensions of BHK21 tissue culture fibroblasts

BHK21 hamster tissue culture fibroblasts, when brought into suspension with trypsin, aggregate spontaneously in serum-free medium. The amount of aggregation appears to depend on the proportion of quiescent (growth inhibited) cells which were present in the culture. It is greatest in cells from cultures whose growth is inhibited, either by high cell density or by low serum concentration. When serum is added to low serum cultures, growth is induced and S phase and mitosis follow at 14 and 20 h respectively. It has now been found that aggregation ceases less than 2 h after the addition of serum. Growth can also be induced in such cultures by infection with high multiplicities of polyoma virus. Infected cells enter S phase about 28 h after infection. It has been found that aggregation ceases between 12 and 20 h after infection. Thus in both these cases loss of aggregation precedes S phase by about 12 h. It is concluded that the absence of aggregation in suspensions derived from polyoma virus-transformed lines of BHK21 cells is a consequence of their resistance to density-dependent inhibition of growth. Failure to undergo spontaneous aggregation appears to be an indicator of an early surface change associated with the induction of growth.     

Cultured Nb rat lymphoma cells in endocrine and cancer research

This review outlines the establishment, properties, and use of two lines of cultured Nb rat lymphoma cells. The cultured cells have retained important properties of the cancers of origin, such as dependency on prolactin for growth and a high sensitivity to antineoplastic Vinca alkaloids. The cultures have been useful for defining the hormonal dependency of the lymphomas in the animal and for studying the progression of the lymphomas from hormonal dependency to autonomy. A new, specific and highly sensitive in vitro bioassay for lactogenic hormones has been developed from one of the cultures. The use of the lymphoma cell cultures has revealed unsuspected pharmacological differences between the closely related, clinically useful antineoplastic Vinca alkaloids, vinblastine and vincristine. The lymphoma cell cultures are also useful tools for studying biochemical, cell cycle related events which follow the mitogenic stimulation of cells by a polypeptide growth factor.     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

Pyruvate accumulation in growth-inhibited cultures of Aerobacter aerogenes

1. Accumulation of pyruvate occurs during the early stages of exponential growth of aerobic, anaerobic and static cultures of a strain of Aerobacter aerogenes. In normal cultures of this organism the content of pyruvate increases until most of the glucose of the medium has been consumed, and then declines rapidly. The presence of unconsumed sugar is not the sole reason for the accumulation of keto acid, since this is unaffected by the addition of extra glucose to either exponentialphase or stationary-phase cultures. 2. In aminopterin-inhibited cultures, the rate of glucose utilization is decreased greatly, and pyruvate continues to accumulate throughout the period of incubation. This prolonged phase of accumulation appears to be a consequence of the growth inhibition, and not to a specific action of aminopterin on the phosphoroclastic breakdown of pyruvate, since it occurs also when growth is restricted by the antibiotics streptomycin, chloramphenicol and neomycin. 3. A possible explanation is suggested for the accumulation of pyruvate in the inhibited cultures.     

A simple feedback control of Escherichia coli growth for recombinant aldolase production in fed-batch mode

Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression.     

Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae

Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been reported to grow in batch cultures on glucose-containing complex media, but not on defined glucose-containing media. By a combination of batch and chemostat experiments it is demonstrated that even in complex media, Pdc- S. cerevisiae does not exhibit prolonged growth on glucose. Pdc- strains do grow in carbon-limited cultures on defined media containing glucose-acetate mixtures. The acetate requirement for glucose-limited growth, estimated experimentally by continuously decreasing the acetate feed to chemostat cultures, matched the theoretical acetyl-CoA requirement for lipid and lysine synthesis, consistent with the proposed role of pyruvate decarboxylase in the synthesis of cytosolic acetyl-CoA.