Oxygen gradients in animal-cell bioreactors

An estimation is made of oxygen gradients in animal-cell bioreactors, using straightforward engineering calculations. Three types of bioreactor are considered: stirred vessel, bubble column and air lift, of sizes between 0.01 and 10 m³. First, the gradient is estimated in the stagnant layer surrounding a cell (15 m), a microcarrier (185 m) with 300 cells attached to it, a macroporous support (1.25 mm) containing 185,00 cells and one (6 mm) containing 4.25 million cells. It is assumed that oxygen consumption is 10^–16 mole O₂·cell^–1·s^–1, while mass transfer coefficients are obtained from Sherwood relations. Circulation and liquid-retention times of the bioreactors are compared with the oxygen-exhaust times of suspensions with 10¹², 10¹³ and 10¹⁴ cells/m³ to estimate if oxygen gradients are likely to exist in the bulk-liquid phase. Finally, the gradient in the liquid film surrounding air bubbles is estimated using k _l A-values obtained from empirical correlations. It is clear from all these estimations that in many situations severe gradients can be expected. The question remains, however, whether gradients should be avoided as much as possible, or may be tolerated to a certain extent or even created on purpose because of possible beneficial effects.     

Scale up aspects of sparged insect-cell bioreactors

Conclusion In this chapter we have attempted to evaluate the most important parameters which can be useful for the pur-pose of design and scale up. Insect cells and animal cells in general can be grown well in large vessels. However, none of the theories and parameters discussed in this chapter have been validated on a larger scale than laboratory and small pilot reactors. Selection of the most suitable design and scale-up method there-fore needs in particular studies in larger vessels. The Kolmogorov theory and the killing-volume model are in this respect the most promising approaches for the optimal design of large-scale animal-cell bioreactors.     

Characterization of the localized hydrodynamic shear forces and dissolved oxygen distribution in sparged bioreactors

Detailed, high-resolution numerical simulations of the bubbly flows, used for oxygen delivery and mixing in mammalian cell suspensions, have been performed. The hydrodynamics, shear and normal forces, mass transfer and mass transport from and around individual bubbles and bubble clusters were resolved for different operating conditions, that is, Weber, Morton, and Schmidt numbers. Suspended animal (e.g., mammalian, insect) cells are known to be susceptible to damage potentially leading to cell death, caused by hydrodynamic stresses and oxygen deprivation. Better knowledge of the magnitude of the shear forces and the extent of mixing of the dissolved oxygen in sparged bioreactors can have a significant impact on their future design and optimization. Therefore, the computed liquid-phase velocity fields were used to calculate and compare the local shear in different types of single bubble wakes and in bubble clusters. Oxygen mass transfer and dissolved oxygen transport were resolved to examine oxygen supply to the cells in the different types of flows.     

Hydrodynamic stress and lethal events in sparged microalgae cultures

The effect of high superficial gas velocities in continuous and batch cultures of the strains Dunaliella tertiolecta, Chlamydomonas reinhardtii wild-type and cell wall-lacking mutant was studied in bubble columns. No cell damage was found for D. tertiolecta and C. reinhardtii (wild-type) up to superficial gas velocities of 0.076 and 0.085 m s(-1), respectively, suggesting that high superficial gas velocities alone cannot be responsible for cell death and, consequently, bubble bursting cannot be the sole cause for cell injury. A death rate of 0.46 +/- 0.08 h(-1) was found for C. reinhardtii (cell wall-lacking mutant) at a superficial gas velocity of 0.076 m s(-1), and increased to 1.01 +/- 0.29 h(-1) on increasing superficial gas velocity to 0.085 m s(-1). Shear sensitivity is thus strain-dependent and to some extent the cell wall plays a role in the protection against hydrodynamic shear. When studying the effect of bubble formation at the sparger in batch cultures of D. tertiolecta by varying the number of nozzles, a death rate of 0.047 +/- 0.016 h(-1) was obtained at high gas entrance velocities. D. tertiolecta was cultivated in a pilot-plant reactor under different superficial gas velocities of up to 0.026 m s(-1), with relatively low gas entrance velocities and no cell damage was observed. There is some indication that the main parameter causing cell death and damage was the gas entrance velocity at the sparger.     

Overcoming shear stress of microalgae cultures in sparged photobioreactors

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.     

Reactor design for large scale suspension animal cell culture

The scale of operation of freely suspended animal cell culture has been increasing and in order to meet the demand for recombinant therapeutic products, this increase is likely to continue. The most common reactor types used are stirred tanks. Air lift fermenters are also used, albeit less commonly. No specific guidelines have been published for large scale (≥10 000 L) animal cell culture and reactor designs are often based on those used for microbial systems. However, due to the large difference in energy inputs used for microbial and animal cell systems such designs may be far from optimal. In this review the importance of achieving a balance between mixing, mass transfer and shear effects is emphasised. The implications that meeting this balance has on design of vessels and operation, particularly in terms of strategies to ensure adequate mixing to achieve homogeneity in pH and dissolved gas concentrations are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

Cultivation of hybridoma cells in an inclined bioreactor

Murine hybridoma cells were grown in a bubble column that was inclined up to 45 degrees from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells by rising bubbles and exposure of cells to bursting bubbles were minimized. Maximum viable cell count and exponential growth of the cells were not affected by inclination, but an inclination of 30 degrees gave an antibody titer of 42 mg/L, which more than doubled the yield of 17 mg/L in the vertical position. By comparison, the culture gave yields of 30 mg/L when grown in spinner flasks. The enhanced antibody production in the inclined bioreactor corresponded to a prolonged stationary phase of 45 h. (c) 1995 John Wiley & Sons, Inc.     

Oxygen transfer properties of bubbles in animal cell culture media

Oxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles ( approximately 5 mm diameter) < intermediate bubbles ( approximately 1 mm diameter) < micron-sized bubbles ( approximately 100 mum diameter). Under certain conditions, the micron-sized bubbles were capable of achieving oxygen transfer rate up to 100 h(-1), a 10-20-fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new-born calf serum (NBCS) and/or Pluronic F-68 supplemented media. For the intermediate and micron-sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F-68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron-sized bubbles in Pluronic F-68 supplemented media containing antifoam agent and NBCS.     

气液双升式反应器动物细胞培养及批式生长动力学模型

初步研究了气液双升式动物细胞反应器微载体培养 Bowes细胞和悬浮培养 M4G3杂交瘤细胞的生长条件 ,在不加入消泡剂和保护剂的情况下 ,批式培养 Bowes细胞的最大密度为 2 .6×1 0 6/ml,批式培养 M4G3细胞的最大密度为 1 .5× 1 0 6/ml。基于细胞生长的密度效应 ,建立了动物细胞生长动力学模型 :　　 μ=0　　 t相似文献   

Oxygen transport and consumption by suspended cells in microgravity: a multiphase analysis

A rotating bioreactor for the cell/tissue culture should be operated to obtain sufficient nutrient transfer and avoid damage to the culture materials. Thus, the objective of the present study is to determine the appropriate suspension conditions for the bead/cell distribution and evaluate oxygen transport in the rotating wall vessel (RWV) bioreactor. A numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations. The bead size and rotating speed are the control variables in the calculations. The present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases: 10 rpm for 200 microm; 12 rpm for 300 microm; 14 rpm for 400 microm; 18 rpm for 600 microm. As the rotating speed and the bead size increase from 10 rpm/200 microm to 18 rpm/600 microm, the mean oxygen concentration in the 80% midzone of the vessel is increased by approximately 85% after 1-h rotation due to the high convective flow for 18 rpm/600 microm case as compared to 10 rpm/200 microm case. The present results may serve as criteria to set the operating parameters for a RWV bioreactor, such as the size of beads and the rotating speed, according to the growth of cell aggregates. In addition, it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.     

Xanthan production in bubble column and air-lift reactors

A bubble column (0.05 m(3)) and an air-lift fermentor (1.2 m(3)) were used for the production of the exocellular microbial polysaccharide xanthan with Xanthomonas campestris in a synthetic medium. Upon oxygen depletion in the liquid, the xanthan production rate dropped sharply and then became a linear function of the oxygen transfer rate. The volumetric mass transfer coefficients for oxygen conformed to the correlation of Suh et al. Using this correlation in combination with the model for xanthan batch fermentation suggested by Peters et al., the xanthan fermentations in the bubble column were well described. The model also correctly predicted the time course of the molecular weight of the polysaccharide even when a complex medium was used. In the air-lift fermentor, however, the xanthan production rate and the xanthan yields with respect to oxygen and glucose were lower than expected at the overall oxygen transfer rate. The poor performance of the air lift was traced back to the lack of any oxygen supply in the downcomer.     

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10^–3 m³ liquid per m³ of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10^–4 m³ liquid per m³ of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.     

Quantitative measurement of cell-bubble attachment in bubble columns using foam flotation techniques

A simple and convenient system for quantitatively measuring the number of adsorbed animal cells per unit of bubble surface area (, unit: cells/cm2) was developed. The system was successfully applied to recombinant Chinese hamster ovary (r-CHO) suspension cultures to investigate the dynamic cell-bubble attachment in a bubble column. In serum-free medium, values increased with bubble rising height (H) and cell concentration (C) and then became constant (about 1750 cells/cm2) when H and C were sufficiently high. In medium containing protective additives, the trends of values with H were similar to that in serum-free medium. Compared with serum-free medium, polyvinyl-pyrrolidone (PVP) increased the values to 1941 cell/cm2 whereas other tested additives decreased the values of in some different degree.     

Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling

The supply of oxygen within three-dimensional tissue-engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2-5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 x 10(-9)) + (0.4 x 10(-9) m(2) s(-1)), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 x 10(-10) m(2) s(-1)). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 x 10(-19) mol m(-3) s(-1)), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis.     

Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue

For clinical utility, cardiac grafts should be thick and compact, and contain physiologic density of metabolically active, differentiated cells. This involves the need to control the levels of nutrients, and most critically oxygen, throughout the construct volume. Most culture systems involve diffusional transport within the constructs, a situation associated with gradients of oxygen concentration, cell density, cell viability, and function. The goal of our study was to measure diffusional gradients of oxygen in statically cultured cardiac constructs, and to correlate oxygen gradients to the spatial distributions of cell number and cell viability. Using microelectrodes, we measured oxygen distribution in a disc-shaped constructs (3.6 mm diameter, 1.8 mm thickness) based on neonatal rat cardiomyocytes cultured on collagen scaffolds for 16 days in static dishes. To rationalize experimental data, a mathematical model of oxygen distribution was derived as a function of cell density, viability, and spatial position within the construct. Oxygen concentration and cell viability decreased linearly and the live cell density decreased exponentially with the distance from the construct surface. Physiological density of live cells was present only within the first 128 microm of the construct thickness. Medium flow significantly increased oxygen concentration within the construct, correlating with the improved tissue properties observed for constructs cultured in convectively mixed bioreactors.     

Cell inactivation in the presence of sparging and mechanical agitation

Microbial cells are more readily rendered nonviable by the combined action of air sparging and mechanical agitation than by either action along. A. bubble breakup/coalescence model that incorporates the cell-bubble encounter rate, bubble breakup rate, and death probability is proposed to describe cell inactivation in the presence of bubbles maintained through the joint action of agitation and air, which is continually fed into the impeller stream region via passive vortex entrainment from the surface above or via active sparging from below. Experimental results obtained from a fragile algal (Ochromonas malhamensis) culture are consistent with the model prediction. In particular, the specific cell death rate is linearly related to the specific bubble interfacial surface area. It is shown that cells exhibit sparging-sensitive characteristics when agitation is mild, but become sensitive to surface vortexing when agitation turns vigorous enough to introduce air entrainment. Experimental data obtained from different stirrer sizes are in good agreement with the model (c) 1992 John Wiley & Sons, Inc.     

Nanocarbon Electrocatalysts for Oxygen Reduction in Alkaline Media for Advanced Energy Conversion and Storage

Alkaline oxygen electrocatalysis, targeting anion exchange membrane fuel cells, Zn‐air batteries, and alkaline‐based Li‐air batteries, has become a subject of intensive investigation because of its advantages compared to its acidic counterparts in reaction kinetics and materials stability. However, significant breakthroughs in the design and synthesis of efficient oxygen reduction catalysts from earth‐abundant elements instead of precious metals in alkaline media remain in high demand. Carbon composite materials have been recognized as the most promising because of their reasonable balance between catalytic activity, durability, and cost. In particular, heteroatom (e.g., N, S, B, or P) doping can tune the electronic and geometric properties of carbon, providing more active sites and enhancing the interaction between carbon structure and active sites. Importantly, involvement of transition metals appears to be necessary for achieving high catalytic activity and improved durability by catalyzing carbonization of nitrogen/carbon precursors to form highly graphitized carbon nanostructures with more favorable nitrogen doping. Recently, a synergetic effect was found between the active species in nanocarbon and the loaded oxides/sulfides, resulting in much improved activity. This report focuses on these carbon composite catalysts. Guidance for rational design and synthesis of advanced alkaline ORR catalysts with improved activity and performance durability is also presented.     

On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.