Growth of cells on solid culture medium

Summary For precise experiments with yeast (or other) cells stationary populations are produced by growth on the surface of a solid nutrient medium. The energy supply to these cells is well known from a former publication. The oxygen supply during growth is analysed here in detail. Different types of cell populations can be produced in this way dependent on the thickness of nutrient medium.If such cells are transferred into a liquid buffer solution cell multiplication can be initiated without any nutrient flux into the cell. This new type of initiation of the cell cycle of G₁-cells has to be distinguished from the usual initiation by nutrient supply and from the mechanism of meiotic cell division. The dependence of this cell growth on cell volume, pH-value, oxygen concentration and osmotic pressure is analysed and possibilities to avoid this kind of cell multiplication reaction are discussed.     

Role of nutrient supply on cell growth in bioreactor design for tissue engineering of hematopoietic cells

In the present study, a dynamic mathematical model for the growth of granulocyte progenitor cells in the hematopoietic process is developed based on the principles of diffusion and chemical reaction. This model simulates granulocyte progenitor cell growth and oxygen consumption in a three-dimensional (3-D) perfusion bioreactor. Material balances on cells are coupled to the nutrient balances in 3-D matrices to determine the effects of transport limitations on cell growth. The method of volume averaging is used to formulate the material balances for the cells and the nutrients in the porous matrix containing the cells. All model parameters are obtained from the literature. The maximum cell volume fraction reached when oxygen is depleted in the cell layer at 15 days and is nearly 0.63, corresponding to a cell density of 2.25 x 10(8) cells/mL. The substrate inhibition kinetics for cell growth lead to complex effects with respect to the roles of oxygen concentration and supply by convection and diffusion on cell growth. Variation in the height of the liquid layer above the cell matrix where nutrient supply is introduced affected the relative and absolute amounts of oxygen supply by hydrodynamic flow and by diffusion across a gas permeable FEP membrane. Mass transfer restrictions of the FEP membrane are considerable, and the supply of oxygen by convection is essential to achieve higher levels of cell growth. A maximum growth rate occurs at a specific flow rate. For flow rates higher than this optimal, the high oxygen concentration led to growth inhibition and for lower flow rates growth limitations occur due to insufficient oxygen supply. Because of the nonlinear effects of the autocatalytic substrate inhibition growth kinetics coupled to the convective transport, the rate of growth at this optimal flow rate is higher than that in a corresponding well-mixed reactor where oxygen concentration is set at the maximum indicated by the inhibitory kinetics.     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Individual-based modeling of phytoplankton: evaluating approaches for applying the cell quota model

Present phytoplankton models typically use a population-level (lumped) modeling (PLM) approach that assumes average properties of a population within a control volume. For modern biogeochemical models that formulate growth as a nonlinear function of the internal nutrient (e.g. Droop kinetics), this averaging assumption can introduce a significant error. Individual-based (agent-based) modeling (IBM) does not make the assumption of average properties and therefore constitutes a promising alternative for biogeochemical modeling. This paper explores the hypothesis that the cell quota (Droop) model, which predicts the population-average specific growth or cell division rate, based on the population-average nutrient cell quota, can be applied to individual algal cells and produce the same population-level results. Three models that translate the growth rate calculated using the cell quota model into discrete cell division events are evaluated, including a stochastic model based on the probability of cell division, a deterministic model based on the maturation velocity and fraction of the cell cycle completed (maturity fraction), and a deterministic model based on biomass (carbon) growth and cell size. The division models are integrated into an IBM framework (iAlgae), which combines a lumped system representation of a nutrient with an individual representation of algae. The IBM models are evaluated against a conventional PLM (because that is the traditional approach) and data from a number of steady and unsteady continuous (chemostat) and batch culture laboratory experiments. The stochastic IBM model fails the steady chemostat culture test, because it produces excessive numerical randomness. The deterministic cell cycle IBM model fails the batch culture test, because it has an abrupt drop in cell quota at division, which allows the cell quota to fall below the subsistence quota. The deterministic cell size IBM model reproduces the data and PLM results for all experiments and the model parameters (e.g. maximum specific growth rate, subsistence quota) are the same as those for the PLM. In addition, the model-predicted cell age, size (carbon) and volume distributions are consistent with those derived analytically and compare well to observations. The paper discusses and illustrates scenarios where intra-population variability in natural systems leads to differences between the IBM and PLM models.     

Development of a small-scale bioreactor: application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta). Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH.     

Life‐cycle kinetic model for endospore‐forming bacteria,including germination and sporulation

We develop a mechanistic life‐cycle model for endospore‐forming bacteria (EFB) and test the model with experiments with a Bacillus mixed culture. The model integrates and quantifies how sporulation and germination are triggered by depletion or presence of a limiting substrate, while both substrates affect the rate of vegetative growth by a multiplicative model. Kinetic experiments show the accumulation of small spherical spores after the triggering substrate is depleted, substantially more rapid decay during sporulation than for normal decay of vegetative cells, and a higher specific substrate utilization rate for the germinating cells than that for growth of vegetative cells. Model simulations capture all of these experimental trends. According to model predictions, when a batch reactor is started, seeding with EFB spores instead of active EFB delays the onset of rapid chemical oxygen demand (COD) utilization and biomass growth, but the end points are the same. Simulated results with low aeration intensity show that germination can consume some substrate without dissolved oxygen (DO) depletion. Biotechnol. Bioeng. 2009; 104: 1012–1024. © 2009 Wiley Periodicals, Inc.     

A semicontinuous culture model that links cell growth to extracellular nutrient concentration

During semicontinuous culture, a sample of fixed volume is removed at regular time intervals to make measurements and/or harvest culture components, and an equal volume of fresh medium is immediately added to the culture, thereby instantaneously enhancing nutrient concentrations and diluting cell concentration. The resulting cell concentration versus time curve (i.e., the actual cell growth curve) has a saw-toothed appearance because of the periodic dilution of cell concentration. The observed cell concentrations correspond to the peaks of the saw-toothed curve. Cell growth rates are estimated from the locus of observed cell concentrations (i.e., from the apparent growth curve obtained by connecting the peaks of the saw-toothed curve). The sole preexisting model (Fencl's mode) for estimating cell growth rate is valid only when the cells are growing exponentially at a constant rate between samplings. This model has limited validity: despite the periodic enhancement of nutrient concentration, cell growth between samplings eventually causes nutrient depletion, and the cells cease to grow exponentially. Failure to recognize the limits of validity for Fencl' model has resulted in many erroneous applications of the model and, consequently, many incorrect estimates of cell growth rates. To provide a means for correctly estimating cell growth rates, Fencl's exponential model was extended, and a new model that describes the effects of nutrient depletion on cell growth in semi-continuous culture was obtained. The new model shows that exhaustion of a single growth-limiting nutrient in semicontinuous culture causes the locus of cell concentrations observed at time intervals of Deltat to follow a logistic growth curve. The actual cell growth rate was shown to equal the apparent logistic growth rate plus the effective dilution rate -Deltat(-1) In (1 - f), where f is the ratio of sample volume to total culture volume. Moreover, the model predicts that both the apparent logistic growth rate and the apparent steady-state cell concentration should rise linearly with the concentration of growth-limiting nutrient in the input medium, but fall linearly with increases in the effective dilution rate. The new logistic model for nutrient-limited cell growth in semicontinuous culture was successfully tested using published data for Asterionella formosa, Cyclotella meneghiniana, Daucus carota, and strain L mouse cells.     

Monoclonal antibody productivity and the metabolic pattern of perfusion cultures under varying oxygen tensions

The metabolic pattern and cell culture kinetics of high-cell-density perfusion cultures were compared under two different oxygen transfer conditions: oxygen limiting and not limiting. When oxygen was a limiting factor during perfusion culture, both specific glucose uptake and lactate production rates increased, compared to non-oxygen-limited condition, by about 60% and 30%, respectively. The specific glutamine uptake rate under oxygen-limited conditions was almost 4.0 times higher than that under non-oxygen-limited conditions. The activity of lactate dehydrogenase (LDH) released into the medium by the dead cells can be used as an indicator for the metabolic and physiological conditions related to oxygen limitation. There was a 3.2 times higher specific rate of LDH activity released by dead cells in oxygen-limited cultures than those in non-oxygen-limited cultures. The specific production rate of monoclonal antibody was not significantly affected by the oxygen transfer conditions during the rapid cell growth period, but it rapidly increased toward the end of perfusion cultures. The higher perfusion rate may have limited further cell growth during high-cell-density perfusion culture, because cell damage was caused by the hydrodynamic shear within a hollow fiber microfiltration cartridge installed to withdraw the spent medium and the waste metabolites. (c) 1993 John Wiley & Sons, Inc.     

A Microscope Automated Fluidic System to Study Bacterial Processes in Real Time

Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.     

The cellular-induced decay of DMPO spin adducts of .OH and .O2

In a recent report, it was concluded that DMPO, often considered the spin trap of choice for detection of superoxide and hydroxyl radical adducts in biological systems, may be unsuitable for many biological uses because of its instability in cellular systems. It was demonstrated in red blood cells and in hamster V79 cells that the DMPO spin adducts of .O2- and .OH are metabolized very rapidly so that even if formed, they may not be detected in many experiments with cells. Because of the potential importance of these findings to experiments already reported on the occurrence of oxygen radicals in cellular systems, and the implications of these findings for future experiments, we have extended the studies on DMPO to other cellular, systems. We have also investigated the role of oxygen in this system because it has been shown recently that very hypoxic cells reduce some nitroxides much more rapidly than oxic cells and therefore it seemed possible that the rapid loss of radical adducts of DMPO was due to the hypoxic conditions under which the previous experiments were carried out. The results of the present experiments indicate that the loss of the DMPO spin adducts occurs in other cell systems as well, that the decomposition rate is independent of the concentration of oxygen, and that the final products of cellular metabolism of DMPO adducts are different from those of most nitroxides. There is no evidence that intracellular DMPO-spin adducts of oxygen radicals can be observed under conditions similar to those used in this study. We conclude that DMPO is not likely to be a suitable agent for studying intracellular oxygen radicals.     

A hollow-fiber reactor design for NMR studies of microbial cells

A hollow-fiber membrane reactor was designed and constructed to allow perfusion of entrapped, dense Escherichia coli cells with nutrient medium during examination of cell metabolism using nuclear magnetic resonance (NMR) spectroscopy. Phosphorus-31 NMR spectra of the perfused cells included peaks for nucleoside di- and triphosphates, sugar phosphates, and pH-sensitive peaks for inorganic phosphate. The observed intensity of the lumenal inorganic phosphate peak was found to depend on flow rate, ruling out the use of this peak as a concentration reference. Absolute intracellular pH values obtained from NMR measurements were found to be accurate to 0.2 pH units due to uncertainties in intracellular ionic concentrations. Relative pH values, however, were found to be sensitive to cell energetic status. The response of E. coli intracellular pH following a shift to carbon starvation medium was monitored with a resolution of 3 min. Use of a hollow-fiber reactor for cell containment and perfusion during NMR spectroscopy enables metabolic experiments of longer duration and of greater variety than is possible using standard, nonperfused sample tubes.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

The Role of Boron in Plant Growth: II. THE EFFECT ON GROWTH OF THE RADICLE

Estimates of cell number, cell volume, respiratory rate, nitrogen,sugar, and nucleic acid content were made on 1 mm. sectionsof the radicles of field bean at frequent intervals during thefirst 96 hours of growth in nutrient solutions with and withoutboron. The primary effects of deficiency were cessation of cell divisionand enlargement of the apical cells. The increased volume ofthe apical cells may have been due to either a longer periodbeing available for development as the rate of mitosis decreased,or to an unusually rapid rate of cell extension. The resultsindicated that cell division did not cease for lack of availablesugar nor as a result of failure to synthesize protein or nucleicacids. It is suggested that in the absence of boron divisionceases because abnormalities in the formation of the cell wallprevent the cell from becoming organized for mitosis. In particularthe hypothesis that boron is concerned with the formation of‘pectin’ from uridinediphosphate-D-glucose is examinedin the light of published evidence.     

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.     

Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.     

Hybrid cellular automaton modeling of nutrient modulated cell growth in tissue engineering constructs

Mathematic models help interpret experimental results and accelerate tissue engineering developments. We develop in this paper a hybrid cellular automata model that combines the differential nutrient transport equation to investigate the nutrient limited cell construct development for cartilage tissue engineering. Individual cell behaviors of migration, contact inhibition and cell collision, coupled with the cell proliferation regulated by oxygen concentration were carefully studied. Simplified two-dimensional simulations were performed. Using this model, we investigated the influence of cell migration speed on the overall cell growth within in vitro cell scaffolds. It was found that intense cell motility can enhance initial cell growth rates. However, since cell growth is also significantly modulated by the nutrient contents, intense cell motility with conventional uniform cell seeding method may lead to declined cell growth in the final time because concentrated cell population has been growing around the scaffold periphery to block the nutrient transport from outside culture media. Therefore, homogeneous cell seeding may not be a good way of gaining large and uniform cell densities for the final results. We then compared cell growth in scaffolds with various seeding modes, and proposed a seeding mode with cells initially residing in the middle area of the scaffold that may efficiently reduce the nutrient blockage and result in a better cell amount and uniform cell distribution for tissue engineering construct developments.     

Light as an Energy Source in Continuous Cultures of Bacteriorhodopsin-Containing Halobacteria

The role of light as an energy source for slightly aereated cultures of halobacteria was studied, using continuous cultures with low nutrient concentrations and a low oxygen supply. A series of experiments were carried out with non-illuminated and differently illuminated cultures and with different oxygen transfer rates. Under low oxygen availability, light proved to be a decisively important energy source that allowed the populations to reach higher growth rates and much higher population densities. Oxygen influenced the growth over only a minimal level, below which neither the illuminated nor the dark cultures were affected by the oxygen transfer rate. From these results, it appears that the bacteriorhodopsin-mediated energy supply could have a very important role for the ecology of halobacteria in their microaerophilic habitats. In the illuminated cultures, cells that originated purple colonies on plates appeared. These cells, which could be bacteriorhodopsin-constitutive mutants, are now being studied.     

A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multicellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 micron. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 micron, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid-derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.