Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!

Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodinium cohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 × 10⁶ W/m³ with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale‐up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

On-line measurement of oxygen uptake in cell culture using the dynamic method

Measurement of oxygen uptake rate is useful in assessing growth, viability, and metabolic activity. In cell culture, however, the oxygen demand is extremely small (typically 0.1-0.3 mM O(2)L-h) and is very difficult to measure accurately using conventional offgas analysis. In many industrial submerged cell culture systems, dissolved oxygen levels are controlled between preset limits by intermittent sparging of air or oxygen. This article describes a computational method for the automatic online determination of oxygen uptake from the dynamic dissolved oxygen probe response. Experimental measurements show that for a typical hybridoma culture, specific oxygen demand is 0.15 mM O(2)/10(9) cells/h. (c) 1996 John Wiley & Sons, Inc.     

Studies on Oxygen Transfer in Submerged Fermentations

In conventional shaken culture system, control of oxygen supply is performed by changing liquid volume in flasks and it necessarily introduces variation in the effectiveness of agitation and in the partial pressure of carbon dioxide. In jar or tank culture system, also, the changes in mechanical agitation and in the flow rate of air for control of aeration induce similar problems. It is impossible, therefore, to isolate the effects of oxygen on microbial metabolism from these accompanying ones. Hence, there is a basic requirement of making clear distinction among them, and in this paper the effects of agitation and carbon dioxide on product formation are presented in glutamic acid fermentation using the apparatus of controlling the level of dissolved oxygen throughout the fermentation.

To obtain fundamental knowledge required for attaining adequate aeration, the rate of oxygen demand in glutamic acid fermentation was discussed in connection with its fermentation rates. On the basis of specific rates, rates of change per unit mass of cells, glutamic acid fermentation was found to fall in the process pattern of Gaden’s type II, in which a constant rate of oxygen demand was sustained for a considerable time. On the basis of volumetric rates, rates of change per unit volume of broths, oxygen demand was recognized to be correlated with growth, sugar utilization and product formation, and it was pointed out particularly that the oxygen demand was closedly related with sugar utilization. In the particular cases where rapid utilization of sugar occurred, therefore, oxygen deficiency was liable to be evoked being unable to fill the growing oxygen demand. This finding might be useful for scale-up studies or process design.     

Batch, fed-batch, and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.     

The cultivation of animal cells at controlled dissolved oxygen partial pressure. Reprinted from Biotechnology and Bioengineering Vol. X, Issue 6, Pages 801-814 (1968)

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO(2)). The culture technique allows metabolically produced CO(2) to be measured; provision can be made to control the dissolved CO(2) partial pressure. In cultures containing a low serum concentration, gas sparging to control pO(2) was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO(2) control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO(2) was controlled at values within the range 40-100 mm Hg.     

The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors

Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome. A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials. A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments. A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK. These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate. The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system. Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells. Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in. (2.54 cm) square silicon chip. With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model. A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices.     

Superoxide dismutase activities in Rhizobium phaseoli bacteria and bacteroids

Superoxide dismutase activity in free-living Rhizobium phaseoli is due to the presence of two different enzymes containing manganese or iron. Under usual culture conditions, the manganese-enzyme appears largely predominant but the induction of the iron-superoxide dismutase can be obtained by addition of methyl viologen to the culture media. The corresponding bacteroid, extracted from French-bean nodules, contains only a manganese-superoxide dismutase whose characteristics are similar to those of the bacterial enzyme. However, the activity of the microsymbiont is slightly lower than that of free-living cells. The presence of an active superoxide dismutase in the bacteroids suggests a significant formation of superoxide anion by their metabolism; this can be correlated with the existence of a large oxygen demand by the microsymbionts within the nodule, as suggested by their important oxygen uptake in vitro.     

Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network

A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc.     

Homogeneous cultivation of animal cells for the production of virus and virus products

Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 10⁷ plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.     

The cultivation of animal cells at controlled dissolved oxygen partial pressure

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO₂). The culture technique allows metabolically produced CO₂ to be measured; provision can be made to control the dissolved CO₂ partial pressure. In cultures containing a low serum concentration, gas sparging to control pO₂ was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO₂ control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO₂ was controlled at values within the range 40–100 mm Hg.     

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。     

Disposable bioreactor for cell culture using wave-induced agitation

This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of endothelial cells in the proliferative response of cultured pulmonary vascular smooth muscle cells to reduced oxygen tension

Summary The development of pulmonary hypertension in a wide variety of human disease states and experimental animal models characterized by chronic alveolar hypoxia is mediated by two pathologic vascular processes, a) vasoconstriction and b) vasoconstruction (structural remodeling). The anatomic changes seen within the pulmonary circulation include a) increased deposition of collagen and elastin in the adventitial layer and b) aberrant pulmonary vascular smooth muscle cell proliferation and maturation in the medial segments. Despite the demonstrated ability of pharmacologic manipulation in the experimental animal to ameliorate both the structural and hemodynamic changes, the actual etiologic mechanisms are only beginning to be explored. Using the cell culture technique of co-cultivation, we have investigated the potential role of bovine pulmonary arterial endothelial cell-derived factors in mediating abnormal bovine smooth muscle cell growth under conditions of reduced oxygen tension. We have demonstrated that these cultured endothelial cells exposed in vitro to reduced levels of atmospheric oxygen concentrations of 5.0% and 2.5% O₂ for durations of 24 to 72 h produce and secrete soluble growth factor(s) which stimulate smooth muscle cell proliferation when compared to cells maintained under standard tissue culture oxygen conditions of 95% room air. This growth-stimulatory effect required the concomitant presence of serum factors (0.5% fetal bovine serum), was inhibited by heparin, was distinct from platelet-derived growth factor, and seemed to have a molecular weight greater than 14 000 Da. We conclude that reduced levels of oxygen tension in vitro can selectively induce pulmonary arterial endothelial cells to release mitogen(s) which can stimulate vascular smooth muscle replication. Furthermore, we speculate that this in vitro finding may be of importance as an etiologic mechanism to explain the accelerated smooth muscle cell growth characteristic of hypoxic pulmonary arteriopathy.     

Scalable inoculation strategies for microcarrier-based animal cell bioprocesses

Scalability is a major demand for high-yield, stable bioprocess systems in animal cell culture-based biopharmaceutical production. Increased yields can be achieved through high-density cell culture, such as in the combination of microcarrier and fluidized bed bioreactor technology. To minimize inocula volume in industrial applications of fluidized bed fermentation systems, it is crucial to increase the bed volume in the reactor during the fermentation process. We tested scale-up strategy for the production of recombinant human arylsulfatase B (ASB) enzyme used in enzyme replacement therapy in patients afflicted with mucopolysaccharidosis type VI (MPS VI). This enzyme was derived from Chinese hamster ovary (CHO) cells cultivated as adherent cell culture on Cytoline macroporous microcarriers (Amersham Biosciences, Uppsala, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR; Amersham Biosciences, Vogelbusch, Austria). Both 1:2 expansion (herein referred to as the addition of fresh, not-yet-colonized microcarriers) and 1:6 expansion of the carrier bed were performed successfully; the cells restarted to proliferate for colonizing these newly added carriers; and the stability of the culture was not negatively affected.     

Mini-scale bioprocessing systems for highly parallel animal cell cultures

Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.     

动物细胞流加培养的技术现状和发展趋势

流加培养是当前重组蛋白生产的主流培养模式。流加式操作主要是根据细胞对营养物质的不断消耗和需求,设计连续或半连续的流加浓缩营养物,使细胞持续高密度的生长,提高单位反应器体积内目的蛋白产量,从而达到高效生产的目的。流加培养工艺的关键技术主要包培养基的优化设计、流加策略的选择及优化、细胞代谢的调控。     

动物细胞培养用多孔载体的研制

多孔载体是一种新型的用于动物细胞培养的优秀的细胞支持物,其内部网状结构的小孔具有固定细胞和保护细胞免受机械损伤的功能,适合于贴壁细胞和悬浮细胞的培养,能提高培养密度,可应用于大规模培养系统。本文综述了多孔载体的物化性质、制作材料和制备方法。     

The Wellcome Foundation Lecture, 1980: monoclonal antibodies from hybrid myelomas

When the lymphoid cells from immunized animals are fused with myeloma cells adapted to grow permanently in culture, hybrid cells can be isolated that are capable of permanent growth in culture, or as transplantable myeloma tumour in animals, and that at the same time express the antibodies of the immunized donor. Such hybrid cells can be cloned and the antibody produced by each clone is monoclonal. By this procedure therefore it is possible to dissect the heterogeneous immune response of an animal. The monoclonal antibodies can be permanently produced in unlimited quantities and the products are well defined chemical entities, unlike antibodies prepared in animals, which vary from animal to animal and even in different periods within a single animal. These properties have been of great importance in the use of antibodies as biochemical reagents in basic research in a variety of fields. They are also replacing conventional antibodies in standard laboratory practice.     

Pilot scale exponential growth of Escherichia coli W to high cell concentration with temperature variation

An efficient method to grow Escherichia coli W to high cell concentrations on the pilot scale is described and discussed. The method involves growth linked introduction of glucose; and ammonia to the culture, sparing with oxygen, and maintenance of aerobic conditions by gradually decreasing the temperature in the culture in order to keep the oxygen demand within the limits of the capacity of supply. Under these conditions the linear rate of cell mass production is actually the result of exponential growth with a gradually decreasing growth-rate constant. About 10 kg packed cells were produced in a 50 liter working-volume fermentor in one run of 13 hr. The concentration of the cells at the end of the growth was about 47 g dry cells/liter. The expenditure for nutrients was minimal and the controls were of simple automatic nature. From the determined yield constants for glucose, nitrogen, phosphorus, and oxygen it may be inferred that the cells grown by this method are similar to those grown exponentially at constant temperature.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.