An automatic system for the assessment of complex medium additives under cultivation conditions

Complex medium additives such as yeast extract or peptone are often used in industrial cell culture processes to prolong cell growth and/or to improve product formation. The quality of those supplements is dependent on the preparation method and can differ from lot to lot. To guarantee consistent production these different lots have to be tested prior to use in fermentation processes. Because a detailed qualitative and quantitative analysis of all components of such a complex mixture is a very difficult task, another assessment method has to be chosen. The best way to evaluate the effect of such supplements is to monitor cell activity during real cultivation conditions with and without the added supplement lot. A bioreactor-based test system has been developed to determine the oxygen requirement of the cells as a response to the addition of a supplement to be tested under standardized conditions. Investigations were performed with a mouse-mouse hybridoma cell line and yeast extracts as an example for complex medium additives. The results showed differences in the impact between different extract lots and between different concentrations of an extract.     

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.     

提高动物乳腺生物反应器表达水平的策略

在转基因动物乳腺生物反应器研究中,组织特异性表达水平、表达率和表达定位性是决定性的指标。因此,高效表达乳腺生物反应器的制备是许多研究者要实现的目标,也是走向产业化的基本要求。本文从基因构件、动物基因转移方面叙述了如何提高乳腺生物反应器表达水平、增强表达特异性及表达率,着重探讨如何提高动物乳腺生物反应器表达水平。     

A versatile modular bioreactor platform for Tissue Engineering

Tissue Engineering (TE) bears potential to overcome the persistent shortage of donor organs in transplantation medicine. Additionally, TE products are applied as human test systems in pharmaceutical research to close the gap between animal testing and the administration of drugs to human subjects in clinical trials. However, generating a tissue requires complex culture conditions provided by bioreactors. Currently, the translation of TE technologies into clinical and industrial applications is limited due to a wide range of different tissue‐specific, non‐disposable bioreactor systems. To ensure a high level of standardization, a suitable cost‐effectiveness, and a safe graft production, a generic modular bioreactor platform was developed. Functional modules provide robust control of culture processes, e.g. medium transport, gas exchange, heating, or trapping of floating air bubbles. Characterization revealed improved performance of the modules in comparison to traditional cell culture equipment such as incubators, or peristaltic pumps. By combining the modules, a broad range of culture conditions can be achieved. The novel bioreactor platform allows using disposable components and facilitates tissue culture in closed fluidic systems. By sustaining native carotid arteries, engineering a blood vessel, and generating intestinal tissue models according to a previously published protocol the feasibility and performance of the bioreactor platform was demonstrated.     

大规模动物细胞培养技术研究进展

利用动物细胞大规模培养技术可生产多种生物制品,为提高细胞活力和表达水平及有利于表达产物的纯化,采用有多种添加成分的无血清培养基培养细胞,选择更有利于细胞生长又可提高培养细胞密度的微载体和条件温和、易操作、气体交换速度快的生物反应器,在线监控细胞生存环境和生理活动,减少培养过程培养基中的抑制因素,可创造更适合细胞生存的环境,提高表达水平,向细胞中导入抗凋亡基因,可提高细胞活性和蛋白产量。利用多也微载体以球转球方式大规模培养动物细胞有很好的发展前景。     

A novel membrane bioreactor for detoxifying industrial wastewater: I. Biodegradation of phenol in a synthetically concocted wastewater

A novel process has been used to biodegrade phenol present in an acidic (1 M HCI) and salty (5% w/w NaCl) synthetically bioreactor, in which the phenol present in the wastewater is separated from the inorganic components by means of a silicone rubber membrane. Transfer of the phenol from the wastewater and into a biological growth medium allows biodegradation to proceed under controlled conditions which are unaffected by the hostile inorganic composition of the wastewater. At a wastewater flow rate of 18 mL h(-1) (contact time 6 h), 98.5% of the phenol present in the wastewater at an inlet concentration of 1000 mg ( (-1) ) was degraded; at a contact time of 1.9 h, 65% of the phenol was degraded. Phenol degradation was accompanied by growth of a biofilm on the membrane tubes and by conversion of approximately 80% of the carbon entering the system to CO(2) carbon. Analysis of the transport of phenol across the membrane revealed that the major resistance to mass transfer arose in the diffusion of phenol across the silicone rubber membrane. A mathematical model was used to describe the transfer of phenol across the membrane and the subsequent diffusion and reaction of phenol in the biofilm attached to the membrane tube. This analysis showed that (a) the attached biofilm significantly lowers the mass transfer driving force for phenol across the membrane, and (b) oxygen concentration limits the phenol degradation rate in the biofilm. These conclusions from the model are consistent with the experimental results. (c) 1993 Wiley & Sons, Inc.     

Wireless bioreactor for anaerobic cultivation of bacteria

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.     

Three‐dimensional neural differentiation of embryonic stem cells with ACM induction in microfibrous matrices in bioreactors

The clinical use of pluripotent stem cell (PSC)‐derived neural cells requires an efficient differentiation process for mass production in a bioreactor. Toward this goal, neural differentiation of murine embryonic stem cells (ESCs) in three‐dimensional (3D) polyethylene terephthalate microfibrous matrices was investigated in this study. To streamline the process and provide a platform for process integration, the neural differentiation of ESCs was induced with astrocyte‐conditioned medium without the formation of embryoid bodies, starting from undifferentiated ESC aggregates expanded in a suspension bioreactor. The 3D neural differentiation was able to generate a complex neural network in the matrices. When compared to 2D differentiation, 3D differentiation in microfibrous matrices resulted in a higher percentage of nestin‐positive cells (68% vs. 54%) and upregulated gene expressions of nestin, Nurr1, and tyrosine hydroxylase. High purity of neural differentiation in 3D microfibrous matrix was also demonstrated in a spinner bioreactor with 74% nestin + cells. This study demonstrated the feasibility of a scalable process based on 3D differentiation in microfibrous matrices for the production of ESC‐derived neural cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1013–1022, 2013     

Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: Experiments and hydrodynamic modeling

Shear stress is an important biomechanical parameter in regulating human mesenchymal stem cell (hMSC) construct development. In this study, the biomechanical characteristics of hMSCs within highly porous 3-D poly (ethylene terephthalate) (PET) matrices in a perfusion bioreactor system were analyzed for two flow rates of 0.1 and 1.5 mL/min, respectively over a 20-day culture period. A 1.4 times higher proliferation rate, higher CFU-F formation, and more fibronectin and HSP-47 secretion at day 20 were observed at the flow rate of 0.1 mL/min compared to those at the flow rate of 1.5 mL/min. The higher flow rate of 1.5 mL/min upregulated osteogenic differentiation potential at day 20 as measured by the expression of alkaline phosphatase activity and calcium deposition in the matrix after 14 days osteogenic induction, consistent with those reported in literatures. Mathematical modeling indicated that shear stress existed in the range of 1 x 10(-5) to 1 x 10(-4) Pa in the constructs up to a depth of 70 microm due to flow penetration in the porous constructs. Analysis of oxygen transport in the constructs for the two flow rates yielded oxygen levels significantly higher than those at which cell growth and metabolism are affected (Jiang et al., 1996). This indicates that differences in convective transport have no significant influence on cell growth and metabolism for the range of flow rates studied. These results demonstrate that shear stress is an important microenvironment parameter that regulates hMSC construct development at a range significantly lower than those reported previously in the perfusion system.     

Evaluation of a novel Wave Bioreactor® cellbag for aerobic yeast cultivation

The Wave Bioreactor is widely used in cell culture due to the benefits of disposable technology and ease of use. A novel cellbag was developed featuring a frit sparger to increase the system's oxygen transfer. The purpose of this work was to evaluate the sparged cellbag for yeast cultivation. Oxygen mass transfer studies were conducted in simulated culture medium and the sparged system's maximum oxygen mass transfer coefficient (kLa) was 38 h(-1). These measurements revealed that the sparger was ineffective in increasing the oxygen transfer capacity. Cultures of Saccharomyces cerevisiae were successfully grown in oxygen-blended sparged and oxygen-blended standard cellbags. Under steady state conditions for both cellbag designs, kLa values as high as 60 h(-1) were obtained with no difference in growth characteristics. This is the first report of a successful cultivation of a microbe in a Wave Bioreactor comparing conventional seed expansion in shake flasks and stirred tank bioreactors.