Rotating versus perfusion bioreactor for the culture of engineered vascular constructs based on hyaluronic acid

It is generally accepted that dynamic culture conditions are required for vascular tissue engineering. We compared the effects of two dynamic culture systems, a perfusion and a rotating bioreactor, using tubular constructs based on hyaluronic acid seeded with porcine aortic smooth muscle cells (SMC), that we recently showed to be adequate for the generation of vascular tissue. In perfused constructs mechanical stimulation importantly affected cell morphology, increased the incidence of cell proliferation and reduced apoptosis. However, extracellular matrix deposition, cytoskeletal organization and mechanical properties were poor. In rotated constructs cell proliferation was also higher and apoptosis lower than in static controls. Rotated constructs showed the highest ultimate stress and the lowest elastic modulus. Our data indicate that the rotating bioreactor is more efficient than the perfusion bioreactor and we then suggest that this method can be considered a valid alternative to complex bioreactor systems described in the literature.     

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.     

Design and validation of a dynamic flow perfusion bioreactor for use with compliant tissue engineering scaffolds

In tissue engineering, flow perfusion bioreactors can be used to enhance nutrient diffusion while mechanically stimulating cells to increase matrix production. The goal of this study was to design and validate a dynamic flow perfusion bioreactor for use with compliant scaffolds. Using a non-permanent staining technique, scaffold perfusion was verified for flow rates of 0.1-2.0 mL/min. Flow analysis revealed that steady, pulsatile and oscillatory flow profiles were effectively transferred from the pump to the scaffold. Compared to static culture, bioreactor culture of osteoblast-seeded collagen-GAG scaffolds led to a 27-34% decrease in cell number but stimulated an 800-1200% increase in the production of prostaglandin E(2), an early-stage bone formation marker. This validated flow perfusion bioreactor provides the basis for optimisation of bioreactor culture in tissue engineering applications.     

Accelerating HIV-1 VLP production using stable High Five insect cell pools

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

利用动物乳腺生物反应器生产药用蛋白

动物乳腺生物反应器是利用动物乳腺特异性启动子调控元件指导外源基因在乳腺中特异性表达,并从转基因动物奶液中获取重组蛋白。应用动物乳腺生物反应器生产药用蛋白具有生产方式简单,产量大,蛋白能进行翻译后修饰等优点,是具有广阔前景的生物医药产业。本文仅就动物乳腺生物反应器的建立、检测、目的蛋白的分离纯化以及存在的问题等作一综述。     

A rapid and highly efficient method for transformation of sugarcane callus

Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult. Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars. One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol. An improved protocol for Agrobacterium-mediated transformation of sugarcane is described. Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene. The whole procedure permitted the production of transgenic calli in a short time (1.5 mo). The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants. This protocol may be implemented also for the generation of transgenic plants from other species.     

An efficient and scalable process for helper-dependent adenoviral vector production using polyethylenimine-adenofection

Safety requirements for adenoviral gene therapy protocols have led to the development of the third generation of vectors commonly called helper-dependent adenoviral vectors (HDVs). HDVs have demonstrated a high therapeutic potential; however, the poor efficiency and reliability of the actual production process hampers further large-scale clinical evaluation of this new vector. The current HDV production methods involve a preliminary rescue step through transfection of adherent cell cultures by an HDV plasmid followed by a helper adenovirus (HV) infection. Amplification by serial co-infection of complementary cells allows an increase in the HDV titer. Using a HEK293 FLP/frt cell system in suspension culture, an alternative protocol to the current transfection/infection procedure was evaluated. In this work, the adenofection uses the HDV plasmid linked to the HV with the help of polyethylenimine (PEI) and has shown to outperform standard protocols by producing higher HDV yield. The influence of complex composition on the HDV production was examined by a statistical design. The optimized adenofection and amplification conditions were successively performed to generate HDV at the 3 L bioreactor scale. Following only two serial co-infection passages, up to 1.44 x 10(8) HDV infectious units/mL of culture were generated, which corresponded to 26% of the total particles produced. This production strategy, realized in cell suspension culture, reduced process duration and therefore the probability of vector recombination by introducing a cost-effective transfection protocol, ensuring production of high-quality vector stock.     

Equipment design considerations for large scale cell culture

Equipment design is frequently recognized as a key component in the success of GMP biologics manufacturing, but is not always implemented with full appreciation of the processing implications. In the case of mammalian cell culture, there are some recognized issues and risks that develop when transitioning to a large scale of operation. The developing demand for cell culture production capacity in the biopharmaceutical industry has led to a progressive increase in the scale of operation in the last decade. This review will provide a high level summary of the documented process difficulties unique to serum-free large scale (LS) cell culture, analyze the engineering constraints typical of these processes, and suggest some practical equipment design considerations to enhance the productivity, reliability and operability of such systems under GMP manufacturing conditions. A systems approach will be used to establish a good LS bioreactor design practice, providing a discussion on gas distribution, agitation, vessel design, SIP/CIP and control issues. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tubular bioreactors: case study of bioreactor performance for industrial production and scientific research

Application of bioreactors is dominated by industrial production with the consequence that bioreactors also for scientific purposes are mainly used following an empiric pragmatic approach. For the sake of a breakthrough in biotechnology in general, and especially for advanced process development, a more systematic approach is emphasized here. This methodology in bioreactor performance studies is explained and the meaning clarified in a case study of a new type of tubular bioreactor. The central role of so-called "model bioreactors" in bench-scale applications is illustrated as a powerful contribution to the optimal design of bioreactors in technical scale. Pilot plant data in case of a tubular reactor for the production of ethanol with Zymomonas and biopesticides with Bacillus thuringiensis are presented.     

Large-scale mammalian cell culture: methods, applications and products.

Animal cell cultures are used to generate products of enormous biotechnological value. These systems rely on conventional manufacturing techniques using organisms that are the result of either cell fusions or genetic engineering. A wealth of new techniques has allowed improvements and developments to be made in culture medium composition, cell modification, and bioreactor design and operation. This progress is expected to be commercially exploited as new products reach the market place.     

Scale-up and design of a pilot-plant photobioreactor for the continuous culture of Spirulina platensis

Scale-up of bioreactors has the intrinsic difficulty of establishing a reliable relationship among physical parameters involved in the design of the new bioreactor and the physiology of the cultured cells. This is more critical in those cases where a more complex operation of the bioreactor is needed, such as in photobioreactors. A key issue in the operation of photobioreactors is establishing a quantification for the interaction between external illumination, internal light distribution and cell growth. In this paper an approach to the scale-up of a photobioreactor for the culture of Spirulina platensis, based on a mathematical model describing this interaction, and the operation of a previous reactor 10 times smaller is presented. The paper describes the approach followed in the scale-up, the analysis of different design constraints, the physical realization of the new bioreactor design, innovative use of plastic material walls to improve reactor safety, and finally the corroboration of its satisfactory operation.     

New miniaturized hollow-fiber bioreactor for in vivo like cell culture, cell expansion, and production of cell-derived products

We have developed a miniaturized hollow-fiber bioreactor system for mammalian cell culture with a volume of 1 mL. Cell and medium compartments of the bioreactor are separated by a semipermeable membrane, and oxygenation of the cell compartment is accomplished using an oxygenation membrane. As a result of the geometry of the transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60, and REH were cultivated up to densities of 3.5 x 10(7)/mL without medium change or manipulation of the cells. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with flow rate. Depending on the molecular size cutoff of the membranes used, added growth factors such as GM-CSF, as well as factors secreted from the cells, are retained in the cell compartment for up to 1 week. This new miniaturized hollow-fiber bioreactor offers advantages in tissue engineering by continuous nutrient supply for cells in high density, retention of added or autocrine produced factors, and undisturbed long-term culture in a closed system.     

Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

Towards Self‐Regulated Bioprocessing: A Compact Benchtop Bioreactor System for Monitored and Controlled 3D Cell and Tissue Culture

Bioreactors are crucial tools for the manufacturing of living cell‐based tissue engineered products. However, to reach the market successfully, higher degrees of automation, as well as a decreased footprint still need to be reached. In this study, the use of a benchtop bioreactor for in vitro perfusion culture of scaffold‐based tissue engineering constructs is assessed. A low‐footprint benchtop bioreactor system is designed, comprising a single‐use fluidic components and a bioreactor housing. The bioreactor is operated using an in‐house developed program and the culture environment is monitored by specifically designed sensor ports. A gas‐exchange module is incorporated allowing for heat and mass transfers. Titanium‐based scaffolds are seeded with human periosteum‐derived cells and cultured up to 3 weeks. The benchtop bioreactor constructs are compared to benchmark perfusion systems. Live/Dead stainings, DNA quantifications, glucose consumption, and lactate production assays confirm that the constructs cultured in the benchtop bioreactor grew similarly to the benchmark systems. Manual regulation of the system set points enabled efficient alteration of the culture environment in terms of temperature, pH, and dissolved oxygen. This study provides the necessary basis for the development of low‐footprint, automated, benchtop perfusion bioreactors and enables the implementation of active environment control.     

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。     

Insect cell culture for industrial production of recombinant proteins

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.     

生物技术在传统药材生产中的应用前景

生物技术在传统药材生产中的应用前景刘涤胡之璧（上海中医药大学中药生物工程研究室,上海２０００３２）我国的中药材是一个具有数千年历史的医药宝库,至今仍在中国和许多国家和地区广为使用。传统药材中,８０％为野生资源,但由于盲目挖掘,不仅使野生资源日益减少,而且严重破坏了自然界的生态平衡;人工种植又面临品质退化,农药污染和种子带病等问题。而且,人工种植的药材,活性成分的种类和数量往往因地区及气候不同...     

Successful development of small diameter tissue-engineering vascular vessels by our novel integrally designed pulsatile perfusion-based bioreactor

Small-diameter (<4 mm) vascular constructs are urgently needed for patients requiring replacement of their peripheral vessels. However, successful development of constructs remains a significant challenge. In this study, we successfully developed small-diameter vascular constructs with high patency using our integrally designed computer-controlled bioreactor system. This computer-controlled bioreactor system can confer physiological mechanical stimuli and fluid flow similar to physiological stimuli to the cultured grafts. The medium circulating system optimizes the culture conditions by maintaining fixed concentration of O(2) and CO(2) in the medium flow and constant delivery of nutrients and waste metabolites, as well as eliminates the complicated replacement of culture medium in traditional vascular tissue engineering. Biochemical and mechanical assay of newly developed grafts confirm the feasibility of the bioreactor system for small-diameter vascular engineering. Furthermore, the computer-controlled bioreactor is superior for cultured cell proliferation compared with the traditional non-computer-controlled bioreactor. Specifically, our novel bioreactor system may be a potential alternative for tissue engineering of large-scale small-diameter vascular vessels for clinical use.