生物反应器与药用植物毛状根的大规模培养

利用药用植物毛状根培养生产次生代谢产物具有极大的生产潜力,而开发适合毛状根培养的反应器,又是毛状根生产天然产物工业化的关键。本文系统地介绍了各种用于毛状根培养的生物反应器,对通气搅拌式、气升式、超声雾化式等生物反应器各自的特点及优势进行了详细阐述,进一步讨论了各种反应器对于毛状根生长和次生代谢物积累的影响,并提出药用植物毛状根大规模培养的生物反应器在今后的发展方向。     

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.     

动物细胞培养用生物反应器及相关技术

动物细胞大量培养是生产生物制品的重要途径,它用到的关键设备是生物反应器。根据培养细胞、培养载体、培养液混合方式的不同,生物反应器主要有搅拌式、气升式、中空纤维式、回转式等,其中搅拌式规模最大。回转式是NASA于20世纪90年代中期开发的一种新型生物反应器,被誉为空间生物反应器,可用于组织工程研究。与生物反应器配套的技术主要有灌注、微载体、多孔微球、转入抗凋亡基因等,可以有效地提高细胞密度,增加生物制品产量,提高质量。今后生物反应器研制主要朝两个方向发展:一是,以高密度培养动物细胞生产蛋白质药物为目的,二是以三维培养动物细胞(主要是人类细胞)再生组织或器官为目的。     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

Modeling of effects of nutrient gradients on cell proliferation in microfluidic bioreactor

Precise control of the microenvironment is highly desirable in cell culture to study the cell biology. Microfluidic based bioreactors provide a promising method for the spatial and temporal control of cell growth and stimuli. A three-dimensional nutrient transport model, incorporating the monolayer cell growth model, has been developed to investigate the influence of gradients of oxygen and epidermal growth factor (EGF) on the cell culture in the continuous-flow microchannel bioreactor. Our results demonstrate that applying inlet concentration gradients of oxygen and EGF can induce variations of cell density in the y-direction. It is further found that compared to the oxygen gradients the EGF concentration gradients are more efficient in regulating the cell growth.     

Design of well and groove microchannel bioreactors for cell culture

Microfluidic bioreactors have been shown valuable for various cellular applications. The use of micro-wells/grooves bioreactors, in which micro-topographical features are used to protect sensitive cells from the detrimental effects of fluidic shear stress, is a promising approach to culture sensitive cells in these perfusion microsystems. However, such devices exhibit substantially different fluid dynamics and mass transport characteristics compared to conventional planar microchannel reactors. In order to properly design and optimize these systems, fluid and mass transport issues playing a key role in microscale bioreactors should be adequately addressed. The present work is a parametric study of micro-groove/micro-well microchannel bioreactors. Operation conditions and design parameters were theoretically examined via a numerical model. The complex flow pattern obtained at grooves of various depths was studied and the shear protection factor compared to planar microchannels was evaluated. 3D flow simulations were preformed in order to examine the shear protection factor in micro-wells, which were found to have similar attributes as the grooves. The oxygen mass transport problem, which is coupled to the fluid mechanics problem, was solved for various groove geometries and for several cell types, assuming a defined shear stress limitation. It is shown that by optimizing the groove depth, the groove bioreactor may be used to effectively maximize the number of cells cultured within it or to minimize the oxygen gradient existing in such devices. Moreover, for sensitive cells having a high oxygen demand (e.g., hepatocytes) or low endurance to shear (e.g., human embryonic stem cells), results show that the use of grooves is an enabling technology, since under the same physical conditions the cells cannot be cultured for long periods of time in a planar microchannel. In addition to the theoretical model findings, the culture of human foreskin fibroblasts in groove (30 microm depth) and well bioreactors (35 microm depth) was experimentally examined at various flow rates of medium perfusion and compared to cell culture in regular flat microchannels. It was shown that the wells and the grooves enable a one order of magnitude increase in the maximum perfusion rate compared to planar microchannels. Altogether, the study demonstrates that the proper design and use of microgroove/well bioreactors may be highly beneficial for cell culture assays.     

Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.     

Gaseous concentration gradients in tray type solid state fermentors — Effect on yields and productivities

In aerobic solid state fermentation systems, interaction of mass transfer effects with bioreaction plays an important role on the yields and productivities of the bioreactors. Experimental observations on the oxygen and carbon dioxide concentration gradients in a tray type solid state fermentation system are reported in this paper. Steep gradients are experienced in deep beds making large portions of the bioreactor ineffective. The results are useful in the design of the bioreactor in terms of efficient mass transfer as well as critical thickness of the substrate bed to be used.     

Upstream processes in antibody production: evaluation of critical parameters

The demand for monoclonal antibody for therapeutic and diagnostic applications is rising constantly which puts up a need to bring down the cost of its production. In this context it becomes a prerequisite to improve the efficiency of the existing processes used for monoclonal antibody production. This review describes various upstream processes used for monoclonal antibody production and evaluates critical parameters and efforts which are being made to enhance the efficiency of the process. The upstream technology has tremendously been upgraded from host cells used for manufacturing to bioreactors type and capacity. The host cells used range from microbial, mammalian to plant cells with mammalian cells dominating the scenario. Disposable bioreactors are being promoted for small scale production due to easy adaptation to process validation and flexibility, though they are limited by the scale of production. In this respect Wave bioreactors for suspension culture have been introduced recently. A novel bioreactor for immobilized cells is described which permits an economical and easy alternative to hollow fiber bioreactor at lab scale production. Modification of the cellular machinery to alter their metabolic characteristics has further added to robustness of cells and perks up cell specific productivity. The process parameters including feeding strategies and environmental parameters are being improved and efforts to validate them to get reproducible results are becoming a trend. Online monitoring of the process and product characterization is increasingly gaining importance. In total the advancement of upstream processes have led to the increase in volumetric productivity by 100-fold over last decade and make the monoclonal antibody production more economical and realistic option for therapeutic applications.     

Verification of energy dissipation rate scalability in pilot and production scale bioreactors using computational fluid dynamics

Suspension mammalian cell cultures in aerated stirred tank bioreactors are widely used in the production of monoclonal antibodies. Given that production scale cell culture operations are typically performed in very large bioreactors (≥ 10,000 L), bioreactor scale‐down and scale‐up become crucial in the development of robust cell‐culture processes. For successful scale‐up and scale‐down of cell culture operations, it is important to understand the scale‐dependence of the distribution of the energy dissipation rates in a bioreactor. Computational fluid dynamics (CFD) simulations can provide an additional layer of depth to bioreactor scalability analysis. In this communication, we use CFD analyses of five bioreactor configurations to evaluate energy dissipation rates and Kolmogorov length scale distributions at various scales. The results show that hydrodynamic scalability is achievable as long as major design features (# of baffles, impellers) remain consistent across the scales. Finally, in all configurations, the mean Kolmogorov length scale is substantially higher than the average cell size, indicating that catastrophic cell damage due to mechanical agitation is highly unlikely at all scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:760–764, 2014     

Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.     

Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors

Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.     

A Multi-Paradigm Modeling Framework to Simulate Dynamic Reciprocity in a Bioreactor

Despite numerous technology advances, bioreactors are still mostly utilized as functional black-boxes where trial and error eventually leads to the desirable cellular outcome. Investigators have applied various computational approaches to understand the impact the internal dynamics of such devices has on overall cell growth, but such models cannot provide a comprehensive perspective regarding the system dynamics, due to limitations inherent to the underlying approaches. In this study, a novel multi-paradigm modeling platform capable of simulating the dynamic bidirectional relationship between cells and their microenvironment is presented. Designing the modeling platform entailed combining and coupling fully an agent-based modeling platform with a transport phenomena computational modeling framework. To demonstrate capability, the platform was used to study the impact of bioreactor parameters on the overall cell population behavior and vice versa. In order to achieve this, virtual bioreactors were constructed and seeded. The virtual cells, guided by a set of rules involving the simulated mass transport inside the bioreactor, as well as cell-related probabilistic parameters, were capable of displaying an array of behaviors such as proliferation, migration, chemotaxis and apoptosis. In this way the platform was shown to capture not only the impact of bioreactor transport processes on cellular behavior but also the influence that cellular activity wields on that very same local mass transport, thereby influencing overall cell growth. The platform was validated by simulating cellular chemotaxis in a virtual direct visualization chamber and comparing the simulation with its experimental analogue. The results presented in this paper are in agreement with published models of similar flavor. The modeling platform can be used as a concept selection tool to optimize bioreactor design specifications.     

Design of a tubular loop bioreactor for scale-up and scale-down of fermentation processes

Microorganisms traveling through circulation loops in large-scale bioreactors experience variations in their environment such as dissolved oxygen concentration and pH gradients. The same changes are not experienced in small bioreactors, and it is suggested that herein lies one of the major reasons for the problems encountered when translating fermentation data from one scale to another. One approach to study this problem is to look at the circulation loop itself. The present work concerns an attempt to simulate the circulation loops inside stirred tank reactors, using a tubular loop reactor specially constructed for the purpose. The reactor carries a number of ports and probes along its length for the determination of concentration gradients within. The broth is circulated around the loop by the use of peristaltic pumps, and the circulation time (t(c), s) is used as a measure of simulated reactor size. The reactor system has been evaluated using the citric acid fermentation by Aspergillus niger as a test process. Acid production and fungal morphology, in terms of the mean convex perimeter of mycelial clumps quantified by image analysis, were used as the parameters of evaluation for the two systems in comparison. From comparative experiments carried out in 10 and 200 L stirred tank bioreactors, it appears that the loop reactor simulates the corresponding stirred tank representing a valuable tool in scaling up and scaling down of fermentation process.     

Model-based analysis and design of a microchannel reactor for tissue engineering

Recently developed perfusion micro-bioreactors offer the promise of more physiologic in vitro systems for tissue engineering. Successful application of such bioreactors will require a method to characterize the bioreactor environment required to elicit desired cell function. We present a mathematical model to describe nutrient/growth factor transport and cell growth inside a microchannel bioreactor. Using the model, we first show that the nature of spatial gradients in nutrient concentration can be controlled by both design and operating conditions and are a strong function of cell uptake rates. Next, we extend our model to investigate the spatial distributions of cell-secreted soluble autocrine/paracrine growth factors in the bioreactor. We show that the convective transport associated with the continuous cell culture and possible media recirculation can significantly alter the concentration distribution of the soluble signaling molecules as compared to static culture experiments and hence needs special attention when adapting static culture protocols for the bioreactor. Further, using an unsteady state model, we find that spatial gradients in nutrient/growth factor concentrations can bring about spatial variations in the cell density distribution inside the bioreactor, which can result in lowered working volume of the bioreactor. Finally, we show that the nutrient and spatial limitations can dramatically affect the composition of a co-cultured cell population. Our results are significant for the development, design, and optimization of novel micro-channel systems for tissue engineering.     

Control in bioreactors showing gradients

In large-scale bioreactors gradients often occur as a result of non-ideal mixing. This phenomenon complicates design and control of large-scale bioreactors. Gradients in the oxygen concentration can be modeled with a two-compartment model of the liquid phase. Application of this model had been suggested for the control of the dissolved oxygen concentration with a batch gluconic acid fermentation process as the model system. The control system consists of a controller, an observer and a parameter estimator. In this work, the controller design is reconsidered and, in simulation experiments, the performance of the control system has been investigated. When the parameter values are known, the controller in combination with the observer works adequately. The parameter estimator, however, yields incorrect parameters, which are caused by a coupling between two parameters. This causes a deviation of the estimated states from the process states. The simulation results suggest that a priori knowledge of the parameters is required for application of the model for control and state estimation.     

Species Diversity Improves the Efficiency of Mercury-Reducing Biofilms under Changing Environmental Conditions

Six mercury-resistant environmental proteobacterial isolates and one genetically modified mercury-resistant Pseudomonas putida strain were analyzed for physiological traits of adaptive relevance in an environment of packed-bed bioreactors designed for the decontamination of mercury-polluted chlor-alkali wastewater. The strains displayed characteristic differences in each trait (i.e., biofilm formation capability, growth rate in mercury contaminated wastewaters, and mercury reduction efficiency). Subsequently, they were immobilized either as a monoculture or as a mixed culture on porous carrier material in packed-bed bioreactors through which different batches of filter-sterilized industrial chlor-alkali wastewater were pumped. In monospecies bioreactors, the mercury retention efficiency was sensitive to rapidly increasing mercury concentrations in the wastewater. Mixed culture biofilms displayed a high mercury retention efficiency that was not affected by rapid increases in mercury or continuously high mercury concentrations. The dynamic in the community composition of the mixed culture bioreactors was determined by ribosomal intergenic spacer polymorphism analysis. Mercury-mediated selective pressure decreased the number of prevalent strains. Microbial diversity was completely restored after easing of the selective pressure. Microbial diversity provides a reservoir of strains with complementary ecological niches that results in a superior bioreactor performance under changing environmental conditions.     

Species diversity improves the efficiency of mercury-reducing biofilms under changing environmental conditions

Six mercury-resistant environmental proteobacterial isolates and one genetically modified mercury-resistant Pseudomonas putida strain were analyzed for physiological traits of adaptive relevance in an environment of packed-bed bioreactors designed for the decontamination of mercury-polluted chlor-alkali wastewater. The strains displayed characteristic differences in each trait (i.e., biofilm formation capability, growth rate in mercury contaminated wastewaters, and mercury reduction efficiency). Subsequently, they were immobilized either as a monoculture or as a mixed culture on porous carrier material in packed-bed bioreactors through which different batches of filter-sterilized industrial chlor-alkali wastewater were pumped. In monospecies bioreactors, the mercury retention efficiency was sensitive to rapidly increasing mercury concentrations in the wastewater. Mixed culture biofilms displayed a high mercury retention efficiency that was not affected by rapid increases in mercury or continuously high mercury concentrations. The dynamic in the community composition of the mixed culture bioreactors was determined by ribosomal intergenic spacer polymorphism analysis. Mercury-mediated selective pressure decreased the number of prevalent strains. Microbial diversity was completely restored after easing of the selective pressure. Microbial diversity provides a reservoir of strains with complementary ecological niches that results in a superior bioreactor performance under changing environmental conditions.     

The role of bioreactors in tissue engineering

Ex vivo engineering of living tissues is a rapidly developing area with the potential to impact significantly on a wide-range of biomedical applications. Major obstacles to the generation of functional tissues and their widespread clinical use are related to a limited understanding of the regulatory role of specific physicochemical culture parameters on tissue development, and the high manufacturing costs of the few commercially available engineered tissue products. By enabling reproducible and controlled changes of specific environmental factors, bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In addition, by automating and standardizing tissue manufacture in controlled closed systems, bioreactors could reduce production costs, thus facilitating a wider use of engineered tissues.     

Optimal parametric sensitivity control for the estimation of kinetic parameters in bioreactors

In this paper the well-known problem of optimal input design is considered. In particular, the focus is on input design for the estimation of kinetic parameters in bioreactors. The problem is formulated as follows: given the model structure (f,g), which is assumed to be affine in the input, and the specific parameter of interest theta;(k) find a feedback law that maximizes the sensitivity of the model output to the parameter under different flow conditions in the bioreactor and, possibly, minimize the input or state costs. Analytical solutions to these problems are presented. As an example a bioreactor with a biomass that grows according to the well-known Monod kinetics is considered.