Control of growth and differentiation of bioreactor cultures of Physcomitrella by environmental parameters

The effect of physical and chemical environmental parameters on growth and differentiation of suspension cultures of the moss Physcomitrella patens in bioreactors was investigated. By supplementation of the aeration gas with 2% (v/v) CO₂ as well as by continuous illumination, growth of this photoautotrophic growing batch culture was markedly enhanced, resulting in a doubling time of 1.2d. The growth rate of semicontinuously growing bioreactor cultures was not affected by controlling the pH of the culture medium with set points at 4.5 or 7.0. However, growth of the culture at pH 7.0 resulted in increased caulonema development, thus showing a distinct effect on moss differentiation. The impact on research and plant biotechnological applications of the potential to control moss growth and differentiation by environmental parameters is discussed.     

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4?L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ?=?0.07?day^?1 versus 0.05?day^?1) and growth maximum (G _max, 169.10⁶ versus 41.10⁶ cells L^?1). Maximal gymnodimine concentration (1200?μg L^?1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell^?1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L^?1], though the yield [pg cell^?1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793?μg day^?1 over 58?days in continuous culture was achieved versus an average of 60?μg day^?1 [carboy over 40?days] or 249?μg day^?1 [batch mode] over 26?days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25?days, the yield of SPX-desMe-C (29?μg day^?1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day^?1 and 7.7?μg day^?1 respectively). Similarly 5.4?L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12?μg day^?1 versus 7?μg day^?1). When the bioreactor system was upscaled to 52?L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum

There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO₂ in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO₂ aeration, the growth rate nearly doubled from 0.78 to 1.53 d^?1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO₂ during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.     

Non-invasive methods for determination of cellular growth inPodophyllum hexandrum suspension cultures

Culture conductivity and on-line NADH fluorescence were used to measure cellular growth in plant cell suspension cultures ofPodophyllum hexandrum. An inverse correlation between dry cell weight and medium conductivity was observed during shake flask cultivation. A linear relationship between dry cell weight and culture NADH fluorescence was obtained during the exponential phase of batch cultivation in a bioreactor under the pH stat (pH 6) conditions. It was observed that conductivity measurement were suitable for biomass characterisation under highly dynamic uncontrolled shake flask cultivation conditions. However, if the acid/alkali feeding is done for pH control the conductivity measurement could not be applied. On the other hand the NADH fluorescence measurement allowed online-in situ biomass monitoring of rather heterogenous plant cell suspension cultures in bioreactor even under the most desirable pH stat conditions.     

红豆杉细胞悬浮培养结构化数学模型的探讨

用１０Ｌ机械搅拌式生物反应器悬浮培养红豆杉细胞,得到细胞生长、基质消耗和紫杉醇合成动力学曲线。经过代谢动力学分析建立了结构化数学模型。并将模型值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

Protein-free human-human hybridoma cultures in an intercalated-spiral alternate-dead-ended hollow fiber bioreactor

Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.(c) John Wiley & Sons, Inc.     

Kinetic studies of paclitaxel production by Taxus canadensis cultures in batch and semicontinuous with total cell recycle

Suspension cultures of Taxus canadensis were elicited with methyl jasmonate (MJ) under defined headspace ethylene concentrations. Kinetic studies of growth, nutrient consumption, pH variation, and paclitaxel accumulation were conducted in batch cultures and semicontinuous culture with total cell recycle. A dramatic increase of paclitaxel was obtained when the cultures were elicited with 100 microM MJ, but cell growth was thereby arrested. Supplementation of acetyl-CoA and MJ to the culture proved to be another way to improve paclitaxel yields. Using semicontinuous culture with total cell recycle, paclitaxel accumulation was increased by a factor of 4.0 relative to that in the batch culture during 35 days of cultivation.     

Expansion of mesenchymal stem cells under atmospheric carbon dioxide

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system – which relies upon an elevated CO₂ environment – typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO₂ levels negatively affecting cell growth, but more importantly, the need to sparge CO₂ into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO₂ environment in static cultures. MSC expanded under atmospheric CO₂ with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self‐renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1298–1306, 2013     

红光下培养方式对雨生红球藻细胞增殖及其活力的影响

研究重点针对雨生红球藻绿色游动细胞的增殖培养阶段,分析了在利于细胞增殖的红光条件下,几种培养方式的调整对增殖过程和细胞活力的影响。结果显示:（1）在红光下,增殖平台期维持时间长,细胞活力稳定,细胞中性脂无累积,但进入平台期前,细胞中性脂有规律波动,进入平台期后相对稳定;通过更新率为20%的半连续培养,细胞数产出较批次培养提高57%;半连续培养中细胞呈现胁迫调节的时间较批次培养晚。随着培养时间增加,半连续培养下细胞营养盐吸收能力降低。（2）初始接种密度与细胞增殖速率及细胞光合活力呈负相关:初始密度低的细胞增殖速率较高,细胞光合作用活力高。（3）在培养过程中添加CO2时,最大密度均有提高,达6.0105 cells/mL,较无添加组提高54%;细胞分裂速率均有提高,但红光下较白光下增殖速率高（分别为0.223/d和0.198/d）;添加CO2降低培养液pH,利于维持适宜增殖的pH环境。叶绿素荧光参数以及细胞粒径在红光和白光下有显著差异:红光下,Fv/Fm显著高于白光下;红光下补充CO2显著减小细胞粒径,而白光下粒径无显著变化。研究结果显示,在红光下,采用间断式半连续培养补充CO2培养绿色游动细胞,有利于提升细胞活力与产出。     

Growth and anthocyanin accumulation rates of carrot suspension cultures grown with excess nutrients after semicontinuous culture with different limiting nutrients at several dilution rates, pHs, and temperatures

When carrot cell cultures, after growth in semicontinuous culture, were transferred to media containing excess nutrients, they grew at different rates. The growth rates were generally higher after semicontinuous culture at higher dilution rates. There appears to be a limit on dilution rate above which growth rate does not increase. These changes were also displayed by clones from the parental culture. The possibility that these changes in growth rate reflect a need for the cultures to adapt to their new conditions is discussed. The growth rates of the cultures is markedly increased at 25 degrees C compared with 22 degrees C with little further increase at 28 degrees C. Growth rate is altered by less than 20% when pH is changed from 4.5 to either 5.5 or 4.2. The rates of anthocyanin accumulation by the cultures were similar under all conditions tested except at 22 degrees C. They were larger than the rates of dry weight accumulation. In contrast, the amounts of anthocyanin accumulated in the clones and in the parental cultures grown at pH 5.5 instead of pH 4.5 were very different. The observations were interpreted as showing that the clones differ in the rate of metabolism but not in the rate of synthesis of anthocyanins and that at pH 5.5 the rate of metabolism of anthocyanins but not the rate of synthesis is higher than it is at pH 4.5. The use of semicontinuous cultures as a source of inoculum for batch cultures rather than as a source of biomass for extraction of chemicals is discussed.     

动物细胞培养用生物反应器设计原理

动物细胞培养用生物反应器设计和放大的关键问题是细胞破损与供氧和混合的矛盾,在分析细胞破损机理基础上,提出了动物细胞培养生物反应器的设计原理——设计模型和有关设计条件,从而清楚地确立了细胞死亡速度与培养基组成、反应器设计和操作参数间的定量关系,以及反应器设计应遵循的保证细胞生长和满足传质要求的条件。还对强化传质和抑制细胞破损这一矛盾作了简要分析和讨论。     

Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development

Increasing the throughput and efficiency of cell culture process development has become increasingly important to rapidly screen and optimize cell culture media and process parameters. This study describes the application of a miniaturized bioreactor system as a scaled-down model for cell culture process development using a CHO cell line expressing a recombinant protein. The microbioreactor system (M24) provides non-invasive online monitoring and control capability for process parameters such as pH, dissolved oxygen (DO), and temperature at the individual well level. A systematic evaluation of the M24 for cell culture process applications was successfully completed. Several challenges were initially identified. These included uneven gas distribution in the wells due to system design and lot to lot variability, foaming issues caused by sparging required for active DO control, and pH control limitation under conditions of minimal dissolved CO2. A high degree of variability was found which was addressed by changes in the system design. The foaming issue was resolved by addition of anti-foam, reduction of sparge rate, and elimination of DO control. The pH control limitation was overcome by a single manual liquid base addition. Intra-well reproducibility, as indicated by measurements of process parameters, cell growth, metabolite profiles, protein titer, protein quality, and scale-equivalency between the M24 and 2 L bioreactor cultures were very good. This evaluation has shown feasibility of utilizing the M24 as a scale-down tool for cell culture application development under industrially relevant process conditions.     

Dynamics of single cell property distributions in Chinese hamster ovary cell cultures monitored and controlled with automated flow cytometry

Two important variables that are often not measured online in Chinese hamster ovary (CHO) cell cultures are cell number concentration and culture viability. We have developed an automated flow cytometry system that measured the cell number concentration, single cell viability based on propidium iodide (PI) exclusion, and single cell light scattering from bioreactor samples every 30 min. The bioreactor was monitored during batch growth, and then the cell number concentration was controlled at a set point during cytostat operation. NH₄Cl was added during steady state operation in cytostat mode to monitor the transient cell population response to adverse growth conditions. The automated measurements correlated well to cell concentration and viability determined manually using a hemacytometer. The described system provides a method to study mammalian cell culture physiology and dynamics in great detail. It presents a new method for the monitoring and control of animal cell culture.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999