CFD simulation coupled with population balance equations for aerated stirred bioreactors

Mammalian cells have been widely used to produce therapeutic proteins in stirred bioreactors in suspension culture. Local hydrodynamics can have a great impact on cell proliferation and protein synthesis, but there are few reports on spatial heterogeneity of nutrients, gas bubbles, and mass transfer coefficients. We have employed computational fluid dynamics (CFD) coupled with population balance equations to study local hydrodynamics in a 20 L stirred bioreactor. The flow patterns, energy dissipation rates, gas volume fraction, gas bubble size distribution and local mass transfer coefficient have been displayed throughout the whole bioreactor. Their implications for mammalian cell culture have been discussed. This study provides an insight into rational design and optimum operation conditions in a stirred bioreactor for mammalian cell cultivation.     

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell‐culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell‐carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1002–1012, 2013     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

气升式生物反应器在杂交瘤细胞培养中的应用

前述研究工作基础上,设计开发了10L规模的动物细胞培养用气升式生物反应器。应用该生物反应器悬浮培养杂交瘤细胞．通过平行试验,考察了该反应器设计的合理性和可靠性。结果显示该反应器不存在限制细胞生长、代谢和产物生成的因素,而且细胞破损技彻底消除,表明该气升式生物反应器给细胞生长、代谢和产物生成提供了理想的培养环境,其设计是成功的。     

Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes

Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.     

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor’s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.     

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.     

Comparison of manufacturing techniques for adenovirus production

We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

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。     

机械搅拌式动物细胞反应器不同桨型组合的CFD数值模拟与优化

生物反应器已成为哺乳动物细胞生产治疗性抗体药物和疫苗的核心。文中采用CFD数值模拟方法对目前常用的机械搅拌式生物反应器在不同的搅拌形式下的流场进行了分析,获得了5种搅拌桨型组合条件下的速率矢量、持气率、含气率和剪切力分布的特征。通过构建的重组CHO细胞在不同搅拌形式条件下的流加分批培养发现,细胞密度和抗体表达水平与反应器内的最大剪切率直接相关,在FBMI3搅拌形式下细胞密度和抗体表达水平均最高。结果表明该CHO细胞在悬浮培养时对剪切环境比较敏感,且最大剪切力是工业规模放大的关键因素。     

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

Cost-effective engineering of a small-scale bioreactor

Several methods exist for increasing the scale of cell culture in the laboratory. While these methods provide significant increases in biomass, they are often prohibitively expensive for many laboratories. We have engineered a small-scale bioreactor with a novel means of introducing oxygen through the catalytic breakdown of hydrogen peroxide using a manganese oxide catalyst. We have also adapted and modified an existing assay for dissolved oxygen to be compatible with culture conditions. In this system we have been able to culture CHO cells at densities of up to 10(7) cells/mL without the use of automated feedback systems.     

A simple eccentric stirred tank mini‐bioreactor: Mixing characterization and mammalian cell culture experiments

In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5–100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple—easy to assemble, easy to use, easy to clean—cell culture mini‐bioreactors for lab‐scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini‐bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini‐bioreactor were comparable to those observed for 6‐well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini‐bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini‐bioreactor. Biotechnol. Bioeng. 2013; 110: 1106–1118. © 2012 Wiley Periodicals, Inc.     

Bioreactors for 3-dimensional high-density culture of human cells

A bioreactor was developed as an instrument to culture human or animal cells that require attachment in a large quantity or at a high density. The purpose for developing such a bioreactor is two-fold: to produce a large quantity of animal or human cells that have been modified by gene recombination technology to accommodate manufacture of physiologically-active substances or human proteins on an industrial scale; and for research to culture animal cells to form a high-density 3-dimensional structure as a morphological or functional tissue or organ entity. In the current report, the circulatory flow bioreactor and radial flow bioreactor (RFB) are introduced, in which the former can be scaled up. As a small bioreactor produced for the latter purpose, a rotary cell culture system and novel multicoaxial hollow-fiber bioreactor are introduced. Finally, a small RFB culture system that was scaled down by the present author and his collaborators for the study of a 3-dimensional high density culture system is described. The RFB can be readily scaled up for manufacturing or scaled down for research purposes. This is a cell culturing system that can induce the functions of human tissues by preparing a high density 3-dimensional organization of cells of human origin.     

Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5 years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.     

用填充床生物反应器大规模培养表达重组人红细胞生成素的细胞株

目的：用填充床生物反应器培养表达重组人红细胞生成素的工程细胞株C2W,使其达到高密度高表达。方法：将工程细胞株用含5％小牛血清的DF培养基复苏放大培养,当细胞达到10^9时,接种到5L生物反应器中,先用含血清培养基生长培养,再换为无血清培养基表达培养;在整个培养过程中,采用流加方式连续培养,每日采样测定培养上清中葡萄糖浓度,隔日测定细胞的表达水平。结果：接种量约为10^9细胞;细胞罐培养57d,包括含血清生长培养6d,无血清表达培养51d：重组人红细胞生成素平均表达水平为5636U／mL,最高时达7880U／mL;收集无血清培养上清476L,平均每日灌流量8.3L,最高时达12L／日。结论：在适当的条件下,利用填充床生物反应器可使工程细胞株的培养达到长时间、高表达。     

High-density tissue-like cultivation of JAR choriocarcinoma cells for the in vitro production of human xylosyltransferase

Human xylosyltransferase is the chain-initiating enzyme involved in the biosynthesis of glycosaminoglycans. Large amounts of xylosyltransferase are required to study the biochemical properties of the native enzyme. To achieve this goal a scale-up of animal cell culture systems was inevitable due to the small amounts of the enzyme present in tissues, e.g. only 0.5 microg XT can be obtained from a chick embryo. JAR choriocarcinoma cells cultured with 10% fetal calf serum were found to secrete xylosyltransferase with relatively high activities (1.10 mU l(-1)). To reduce contaminating proteins JAR cells were adapted to serum-free conditions. Xylosyltransferase activities up to 0.22 mU l(-1) were determined in the harvested cell culture supernatant. Scaling-up of JAR cell culture in the hybrid hollow fiber bioreactor Tecnomouse resulted in the production of 15.8 mU or 270 microg XT in 0.5 l of XT-enriched cell culture supernatant using 57 l of serum-free cell culture medium. The XT activity per ml harvest solution was 200-280-fold higher in this cell culture supernatant than in cell culture flasks. In addition, the specific XT activity of the bioreactor product was 6 microU mg(-1) of total protein, which is 2-fold higher than that obtained under static culture conditions. This study clearly demonstrates the successful high-density, tissue-like cultivation of JAR choriocarcinoma cells in a hollow fiber bioreactor resulting in an effective production of native human xylosyltransferase.     

Evaluation of criteria for bioreactor comparison and operation standardization for mammalian cell culture

Development of bioprocesses with mammalian cell culture deals with different bioreactor types and scales. The bioreactors might be intended for generation of cell inoculum and production, research, process development, validation, or transfer purposes. During these activities, not only the difficulty of up and downscaling might lead to failure of consistency in cell growth, but also the use of different bioreactor geometries and operation conditions. In such cases, criteria for bioreactor design and process transfer should be carefully evaluated in order to select appropriate cultivation parameters. In this work, power input, mixing time, impeller tip speed, and Reynolds number have been compared systematically for the cultivation of the human cell line AGE1.HN within three partner laboratories using five different bioreactor systems. Proper operation ranges for the bioreactors were identified using the maximal cell‐specific growth rate (μ_max) as indicator. Common optimum values for process transfer criteria were found in these geometrically different bioreactors, in which deviations of μ_max between cultivation systems can be importantly reduced. The data obtained in this work are used for process standardization and comparability of results obtained in different bioreactor systems, i.e. to guarantee lab‐to‐lab consistency for systems biology approaches using mammalian cells.     

杂交瘤细胞的大量培养

杂交瘤细胞的大量培养是一项迅速发展的技术。本文评述了杂交瘤细胞培养条件和代谢调控方面的研究进展,包括反应器培养中的过程参数优化、细胞损伤和保护、营养物质利用和有害副产物的形成、细胞生长和单抗分泌的动力学以及长期培养的稳定性等问题。同时,本文也讨论了在生物反应器中培养杂交瘤细胞的操作模式和控制策略的研究工作,特别是近年来备受重视的灌注培养和补料培养。