Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.     

Assessment of the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11 during growth by flow cytometry

AIMS: The aim of this study was to improve knowledge about the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11, a chain-forming bacterium, during growth, and to evaluate whether flow cytometry (FCM) combined with fluorescent probes can assess these different physiological states. METHODS AND RESULTS: Cellular viability was assessed using double labelling with carboxyfluorescein diacetate and propidium iodide. FCM makes it possible to discriminate between three cell populations: viable cells, dead cells and cells in an intermediate physiological state. During exponential and stationary phases, the cells in the intermediate physiological state were culturable, whereas this population was no longer culturable at the end of the stationary phase. CONCLUSIONS, AND IMPACT OF THE STUDY: We introduced a new parameter, the ratio of the means of the fluorescence cytometric index to discriminate between viable culturable and viable nonculturable cells. Finally, this work confirms the relevance of FCM combined with two fluorescent stains to evaluate the physiological states of L. lactis SK11 cells during their growth and to distinguish viable cells from viable but not culturable cells.     

Fractal geometry of mosaic pattern demonstrates liver regeneration is a self-similar process.

Partial hepatectomy causes compensatory, nonneoplastic growth and regeneration in mammalian liver. Compensatory liver growth can be used to examine aspects of patterns of cell division in regenerating tissue. Chimeric animals provide markers of cell lineage which are independent of growth and can be used to follow cell division patterns. Previous experimental evidence suggests that compensatory liver growth is uniform, without focal centers of proliferation. In this study we have extended that observation to include genes important in regeneration and cell cycle control in order to establish that nascent growth centers are not present in regenerating liver. There is a uniform spatial distribution of expression of these genes which is not related to mosaic pattern in the chimeras. While these genes may help regulate hepatocyte proliferation they do not appear to regulate patch pattern in the chimeras. With this information confirming uniform growth it was possible to use fractal analysis to test various hypothesized patterns of regenerative growth in the liver. The results of this analysis indicate that mosaic pattern does not change substantially during the regenerative process. Patch area and perimeter (the area occupied by or perimeter around cells of like lineage) increase during compensatory liver growth in chimeric rats without alteration of the geometric complexity of patch boundaries (boundaries around cells of like lineage). These tissue findings are consistent with previously reported computer models of growth in which repetitive application of simple decisions assuming uniform growth created complex mosaic patterns. They support the notion that an iterating (repeating), self-similar (a pattern in which parts are representative of, but not identical to the whole) cell division program is sufficient for the regeneration of liver tissue following partial hepatectomy. Iterating, self-similar cell division programs are important because they suggest a way in which complex patterns (or morphogenesis) can be efficiently created from a small amount of stored information.     

Analysis of protein and cell volume distribution in glucose-limited continuous cultures of budding yeast

Method of flow cytometric analysis have recently been developed that make it possible to obtain segregated data on a single cell basis. In particular, it has been previously demonstrated that protein distributions obtained by flow cytometry give information about the law of growth of the cell population and the law of growth of the single cell; thus these distribution show how the microbial population is actually growing at the moment of the analysis and may yield more accurate and predictive information. We have extended the analysis of protein distribution and cell volume distribution to continuous cultures of Saccharomyces cerevisiae growing in a glucose-limited chemostat. We have found that: (1) to each dilution rate corresponds a given protein and volume distribution that does not change with time in steady state cultures; (2) there is a good proportionality between the average cell volume and the average protein content; (3) the protein distribution obtained can be easily analyzed with the model of growth of yeast previously developed in our laboratory; (4) the analysis of perturbed states shows that both protein distribution and volume distribution change very quickly; thus they are very sensitive parameters and can be used for monitoring and controlling industrial fermentation.     

质谱流式技术用于单细胞检测

质谱流式技术(mass cytometry)是利用质谱原理对单细胞进行多参数检测的流式技术,能够在单细胞水平实现超过50种标志物的同时测量,显著增强了对细胞生长进程和复杂细胞系统的评估能力。该文简要介绍了质谱流式技术的基本工作原理,并从金属元素标记、质量分析器、高维单细胞数据处理等方面展开论述,阐明设计新型金属元素标签和选择飞行时间质谱的必要性,归纳分析高维单细胞数据的算法并总结各种算法的优点和局限性。     

High-density photoautotrophic algal cultures: design, construction, and operation of a novel photobioreactor system

A photobioreactor system has been designed, constructed and implemented to achieve high photosynthetic rates in high-density photoautotrophic algal cell suspensions. This unit is designed for efficient oxygen and biomass production rates, and it also can be used for the production of secreted products. A fiber-optic based optical transmission system that is coupled to an internal light distribution system illuminates the culture volume uniformly, at light intensities of 1.7 mW/cm(2) over a specific surface area of 3.2 cm(2)/cm(3). Uniform light distribution is achieved throughout the reactor without interfering with the flow pattern required to keep the cells in suspension. An on-line ultrafiltration unit exchanges spent with fresh medium, and its use results in very high cell densities, up to 10(9) cells/mL [3% (w/v)] for eukaryotic green alga chlorella vulgaris. DNA histograms obtained form flow cytometric analysis reveal that on-line ultrafiltration influences the growth pattern. Prior to ultrafiltration the cells seem to have at a particular point in the cell cycle where they contain multiple chromosomal equivalents. Following ultrafiltration, these cells divide, and the new cells are committed to division so that cell growth resumes. The Prototype photobioreactor system was operated both in batch and in continuous mode for over 2 months. The measured oxygen production rate of 4-6 mmol/L culture h under continuous operation is consistent with the predicted performance of the unit for the provided light intensity.     

Geometry Shapes Evolution of Early Multicellularity

Organisms have increased in complexity through a series of major evolutionary transitions, in which formerly autonomous entities become parts of a novel higher-level entity. One intriguing feature of the higher-level entity after some major transitions is a division of reproductive labor among its lower-level units in which reproduction is the sole responsibility of a subset of units. Although it can have clear benefits once established, it is unknown how such reproductive division of labor originates. We consider a recent evolution experiment on the yeast Saccharomyces cerevisiae as a unique platform to address the issue of reproductive differentiation during an evolutionary transition in individuality. In the experiment, independent yeast lineages evolved a multicellular “snowflake-like” cluster formed in response to gravity selection. Shortly after the evolution of clusters, the yeast evolved higher rates of cell death. While cell death enables clusters to split apart and form new groups, it also reduces their performance in the face of gravity selection. To understand the selective value of increased cell death, we create a mathematical model of the cellular arrangement within snowflake yeast clusters. The model reveals that the mechanism of cell death and the geometry of the snowflake interact in complex, evolutionarily important ways. We find that the organization of snowflake yeast imposes powerful limitations on the available space for new cell growth. By dying more frequently, cells in clusters avoid encountering space limitations, and, paradoxically, reach higher numbers. In addition, selection for particular group sizes can explain the increased rate of apoptosis both in terms of total cell number and total numbers of collectives. Thus, by considering the geometry of a primitive multicellular organism we can gain insight into the initial emergence of reproductive division of labor during an evolutionary transition in individuality.     

Analysis of bacterial DNA patterns--an approach for controlling biotechnological processes

Optimisation of biotechnological processes catalysed by microbial cells requires detailed information about operational limits of the single cells. Their performance is correlated with distinct physiological states. We related these states to cell cycle events, which were found to proceed extremely diversely in different bacterial strains. Characteristic DNA patterns were found flow cytometrically, depending on the type of strain, substrates and growth conditions involved; this information can be used for the development of control strategies of bioprocesses, although some skill is required.Four bacterial strains (the Gram-negative strains Acinetobacter calcoaceticus 69-V, Ralstonia eutropha JMP 134, Ochrobactrum anthropi K2-14 and the Gram-positive strain Rhodococcus erythropolis K2-3) were grown in mono- and mixed cultures on different substrates, and analysed regarding their proliferation behaviour. The resulting DNA distribution patterns provided three types of valuable information. First, correlation of proliferation activity with the appearance of a major part of cells within the C(2) stage of the cell cycle is a strain-specific feature. Second, bacteria usually maintain more than one chromosome under limiting growth conditions: DNA replication is completed in such cases, but cell division fails. Third, high growth rates are associated with uncoupled DNA synthesis. Its general initiation might be genetically determined in the first place, but it is promoted by optimal growth conditions and the presence of substrates that can be metabolised at high rates, thereby allowing substantial amounts of carbon, other nutrients and energy to be used exclusively for DNA synthesis.     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

Modeling species distributions by breaking the assumption of self-similarity

Species distributions are commonly measured as the number of sites, or geographic grid cells occupied. These data may then be used to model species distributions and to examine patterns in both intraspecific and interspecific distributions. Harte et al. (1999) used a model based on a bisection rule and assuming self-similarity in species distributions to do so. However, this approach has also been criticized for several reasons. Here we show that the self-similarity in species distributions breaks down according to a power relationship with spatial scales, and we therefore adopt a power-scaling assumption for modeling species occupancy distributions. The outcomes of models based on these two assumptions (self-similar and power-scaling) have not previously been compared. Based on Harte's bisection method and an occupancy probability transition model under these two assumptions (self-similar and power-scaling), we compared the scaling pattern of occupancy (also known as the area-of-occupancy) and the spatial distribution of species. The two assumptions of species distribution lead to a relatively similar interspecific occupancy frequency distribution pattern, although the spatial distribution of individual species and the scaling pattern of occupancy differ significantly. The bimodality in occupancy frequency distributions that is common in species communities, is confirmed to a result for certain mathematical and statistical properties of the probability distribution of occupancy. The results thus demonstrate that the use of the bisection method in combination with a power-scaling assumption is more appropriate for modeling species distributions than the use of a self-similarity assumption, particularly at fine scales.     

Surface IgG content of murine hybridomas: Direct evidence for variation of antibody secretion rates during the cell cycle

Previous experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. (c) 1995 John Wiley & Sons Inc.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

Feeding heterogeneity in ciliate populations: Effects of culture age and nutritional state

We have used a novel approach in conjunction with flow cytometry to quantify the biological heterogeneity of populations of the ciliate Tetrahymena pyriformis. It was found that the rate of particle uptake of exponentially growing cells is not uniform among cells and partially correlated with cell size. The physiological state and growth history of the culture was found to affect to a large degree the population's feeding heterogeneity. Stationary phase populations exhibited more uniform feeding behavior, as cell aging affects all cells and effectively reduces their ability to feed. Cells that were removed from the growth medium and resuspended in nonnutritive medium exhibited a more heterogeneous feeding behavior. The starved cells were stimulated to feed at considerably higher rates, and the stimulatory effect was more pronounced for larger cells. It is therefore demonstrated that population heterogeneity has to be evaluated in conjunction with the populations growth state as it is determined by the history of the population's growth and nutritional state. (c) 1994 John Wiley & Sons, Inc.     

The concept of multiple-nutrient-limited growth of microorganisms and its application in biotechnological processes

The "law of the minimum" (Liebig's law) states that usually one nutrient restricts the maximum quantity of biomass that can be produced within a system, whereas all other nutrients are in excess. This general rule has been applied also to the growth of microorganisms, e.g., by adjusting the relative concentrations of the individual nutrients in growth media such that one of them, in the case of heterotrophic microbes, usually the carbon source, determines the maximum cell density that can be obtained in a culture. However, experimental data demonstrated that growth of microbial cultures can be limited simultaneously by two or more nutrients. These authors reported that during growth of bacteria and yeasts at a constant dilution rate in the chemostat, three distinct growth regimes were recognised as a function of the C:N ratio in the inflowing medium: (1) a clearly carbon-limited regime with the nitrogen source in excess, (2) a transition ("double-nutrient-limited") growth regime where both the carbon and the nitrogen source were below the detection limit, and (3) a clearly nitrogen-limited growth regime with the carbon source in excess. Subsequent calculations suggested that the extension and position of this double-nutrient-limited zone should be strongly dependent on the imposed growth rate: Whereas it is very narrow at high growth rates it should become very broad during slow growth. This pattern as a function of growth rate has now been confirmed for a number of different organisms. In industrial processes, microbial growth is always in some way controlled by the limited availability of nutrients, and limitation of specific nutrients is frequently used to force microbial cultures into a productive physiological state. This article will discuss what the consequences of multiple-nutrient-limited growth are for industrial processes and how the concept might be applied. Specific examples will be given that demonstrate the advantages and the potential of multiple nutrient-limited growth conditions for industrial production processes.     

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.     

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model

High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase-plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single-substrate, high-dilution-rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed-substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.     

Immortal DNA or epigenetic signature ?

During mitosis each daughter cell inherits a full copy of the maternal genomic material. DNA replication, however, is an imprecise process, thus errors can arise resulting in potentially deleterious mutations over extended rounds of cell division and these may lead to cancinogenesis. Over thirty years ago, J. Cairns proposed that a cell could avoid the accumulation of mutations arising from DNA replication if all template DNA strands are inherited in one daughter cell during cell division, thus giving rise to the notion of < immortal > DNA strands. In this model the stem cells would retain the template DNA (older) strands. Proving or disproving this notion experimentally has been challenging. Further, it has recently become apparent that epigenetic regulation of gene expression plays a critical role in governing cell states, self-renewal and differentiation. In light of these data, can the phenomenon on template DNA strand segregation also reflect this epigenetic signature? In this review we explore these notions, discuss the evidence in support of this theory, the implications, and some of the mechanisms which could explain this phenomenon.     

Analysis of a cell cycle model based on unequal division of metabolic constituents to daughter cells during cytokinesis

We demonstrate that the unequal division of RNA during cytokinesis explains the dispersion of cell generation times in CHO cell cultures. Experimental cytometric results reported previously serve as a basis for a probabilistic model of cytokinesis. Unequal RNA division to daughter cells, together with two simple laws of RNA production, are used as a source of randomness within the cell cycle. The model reproduces the experimental growth of the CHO cell population, including the observed variability in RNA content. The model has stabilizing properties which explain why a cell population with increased RNA content characteristics, a few cell cycles, to the original pattern. Other cell cycle characteristics, like sister-to-sister and mother-to-daughter generation time correlations implied by the model, are close to their experimental analogs. The conceptual basis of the model is general enough to include unequal division of factors other than RNA (cell mass, cell proteins, etc.) as sources of generation time variability. It seems that the observed dispersion of cell generation times, explained previously in the terms of random transitions in some part of the cell cycle (the Smith & Martin A and B state hypothesis), can be reduced to the single random event of unequal division. This supplies a new convenient tool in the investigation of cell cycle kinetics.     

Determination of cellular rate distributions in microbial cell populations: feeding rates of ciliated protozoa

A novel procedure is proposed for determining distributions of rate properties and correlations of rate with state properties of microbial cell populations. The procedure is novel in that it uses transient data, and thus, it does not require that the population be in balanced growth, although it requires that the population structure does not change during the short transient experiment. The procedure is applied to populations of the ciliated protozoan Tetrahymena to determine ingestion rate variability. The number of ingested microspheres per cell and the single-cell protein content-an indicator of cell size-were directly determined with dual-color flow cytometry. The proposed technique revealed the correlation pattern of the particle ingestion rate with cell size. In particular, ingestion rate was found to be positively correlated with cell size for the smaller feeding cells and to be uncorrelated with size for the larger cells. Using the fact that particle uptake from dilute particle suspensions is a Poisson random process, we determined that the coefficient of variation of the distribution of ingestion rates within the feeding population is about 50%. It was concluded that the dynamics of particle ingestion can be accurately described only if it is realized that particle ingestion rates are distributed. (c) 1993 John Wiley & Sons, Inc.     

Proliferation of dinoflagellates: blooming or bleaching

The dinoflagellates, a diverse sister group of the malaria parasites, are the major agents causing harmful algal blooms and are also the symbiotic algae of corals. Dinoflagellate nuclei differ significantly from other eukaryotic nuclei by having extranuclear spindles, no nucleosomes and enormous genomes in liquid crystal states. These cytological characteristics were related to the acquisition of prokaryotic genes during evolution (hence Mesokaryotes), which may also account for the biochemical diversity and the relatively slow growth rates of dinoflagellates. The fact that the proliferation of many dinoflagellates is sensitive to turbulence may be due to the physiological requirements of the genome's liquid crystal states. Mechanical stress and anti-microtubule drugs induce cell cycle arrest mainly in G1, implicating a role for the permanent cortical microtubular cytoskeleton in mechanotransduction. The cell cycles of photosynthetic dinoflagellates are also gated by the circadian rhythm, with cell division occurring mainly at the end of the dark phase. Cell growth and the biosynthesis of many toxins occur during the light phase, corresponding to G1 in the cell cycle. The dinoflagellates also embody several options for coupling cell cycle progression to cell growth, enabling them to make the best use of available resources and possibly preparing them for a symbiotic existence.