大规模动物细胞固定化培养研究进展

基于固定化载体的生物反应器技术是动物细胞固定化培养的主要手段。综述了近年来微载体,微囊化,无载体细胞自絮凝以及膜等固定化技术在动物细胞固定化培养中的应用及其进展。同时对动物细胞固定化培养中的场效应、多腔室和多细胞混合培养体系进行初步的探讨。     

重组人血白蛋白在哺乳动物细胞培养中的应用

正目前,哺乳动物细胞大规模培养技术已经成为生物制药中不可缺少的一部分,利用动物细胞培养技术生产的生物制品越来越多,而组织工程和干细胞疗法又拓宽了细胞培养技术的应用范围。人血白蛋白(human serum albumin,HSA)作为动物细胞培养基中的重要组分之一,在动物细胞培养中已经应用了很多年。本文就重组人血白蛋白(recombinant HSA,r HSA)在哺乳动物细胞培养中的应用情况作一     

大规模植物细胞培养生物反应器

讨论了植物细胞大规模培养的基本特性。根据分析比较了在植物细胞悬浮培养中通常使用的几种生物反应器,包括摇瓶,搅拌式反应器,气升式反应器,转瓶反应器以及带挡板的转瓶反应器,指出综合性能以气升式为最佳。此外还介绍了固定化植物细胞反应器以及生物膜反应器,讨论了大规模植物细胞培养反应器的选择原则。     

气升式反应器中的西洋参细胞培养

在不同的气升式反应器中对西洋参细胞进行了大规模培养。西洋参细胞得率为11g～14gdwt/e,细胞中总皂甙含量为4.45％。讨论了西洋参细胞在新型的气升式反应器内培养过程中的流变学特性、传质特性以及流场剪切应力变化规律。文章还讨论了培养基成分对大规模培养结果的影响。     

生物反应器无血清悬浮培养MDCK-siat7e细胞工艺的建立

目的建立无血清无蛋白无动物源性培养基BD001悬浮培养MDCK-siat7e细胞的工艺,为MDCK-siat7e细胞的生物反应器规模化培养及病毒性疫苗的研制提供依据。方法采用BD001驯化MDCK-siat7e细胞,建立无血清无蛋白无动物源性MDCK-siat7e细胞库,对细胞库进行常规检定,观察MDCK-siat7e细胞的生长形态;比较不同起始接种密度(4.0×10~5、8.0×10~5、1.6×10~6 cells/ml)悬浮培养,MDCK-siat7e细胞达到平台期的时间、平台期细胞密度及活率;比较不同规模生物反应器(3、15、60 L)无血清无蛋白无动物源性培养MDCK-siat7e细胞对数生长期的比生长速率(μ)、分裂次数(Cd)、葡萄糖比消耗速率(q_(gluc))、乳酸对葡萄糖的转化率(Y_(lac)/g_(luc)),观察细胞的生长和代谢情况。结果建立的无血清无蛋白无动物源性MDCK-siat7e细胞库复苏后细胞平均活率为(96.20±2.95)%。MDCK-siat7e细胞在BD001培养基中复苏传代后生长状态良好,经过24 h潜伏期后,进入对数生长期,第6天进入平台期,第8天达最大细胞密度(6.2×10~6 cells/ml),维持至第12天细胞密度和活率开始下降,进入衰亡期。MDCK-siat7e细胞在无血清培养基中生长良好;不同起始接种密度MDCK-siat7e细胞达到平台期的时间差异有统计学意义(P0.05),平台期细胞密度及活率差异无统计学意义(P0.05);不同规模生物反应器培养的MDCK-siat7e细胞的μ、Cd、q_(gluc)、Y_(lac/gluc)差异均无统计学意义(P0.05),细胞生长代谢良好。结论建立了生物反应器无血清悬浮培养MDCK-siat7e细胞的工艺,为病毒性疫苗生产中采用无血清无蛋白、无动物源性培养基,生物反应器规模化培养MDCK-siat7e细胞提供了一定的实验依据。     

大规模培养动物细胞生产药物

细胞融合与重组ＤＮＡ技术取得的巨大成功为动物细胞培养技术开辟了许多新的领域。目前各类具有明确疗效的生物制品的工业化生产已成为动物细胞培养技术发展的主要动力。动物细胞大规模培养技术的研究开发，除了要解决如何高效地大量生产这些药物之外，其生产过程确保产品质量能适合人体治疗，且不含有害物质，尤其是病毒和ＤＮＡ。     

应用7.5 L生物反应器制备Vero细胞乙型脑炎灭活疫苗

目的应用7.5 L生物反应器,培养Vero细胞和乙型脑炎病毒,规模化生产乙型脑炎灭活疫苗。方法应用7.5 L生物反应器,分别以5、10、15、20、25 g/L的微载体密度加装,采用灌流方式培养Vero细胞,分别将P3株乙型脑炎病毒按0.005 00、0.002 50、0.001 60、0.001 25、0.001 00 MOI接种至生物反应器内,收获乙脑病毒液。经浓缩、灭活、纯化等工艺制备疫苗半成品,经疫苗灭活及纯化试验进行工艺验证,合格后分装为疫苗成品,按照《中国药典》三部(2010版)中《冻干乙型脑炎灭活疫苗(Vero细胞)》要求,进行疫苗全项检定。结果当微载体密度为20、25 g/L时,细胞密度可达1.2×107个/ml,病毒滴度平均达8.33～8.52 lgLD50/ml;当病毒接种量为0.001 25 MOI时,病毒最高滴度可达9.02 lgLD50/ml。经超滤浓缩后的病毒原液,使用1∶4 000β-丙内酯灭活72 h,可达到灭活效果;纯化后可去除90%以上杂蛋白。应用7.5 L生物反应器生产的6批乙型脑炎灭活疫苗,经检定各项指标均符合国家要求。结论应用7.5 L生物反应器培养Vero细胞和P3株乙型脑炎病毒,经连续灌流收获,可规模化生产Vero细胞乙型脑炎灭活疫苗。     

生物反应器培养Vero细胞生产肠道病毒71型

目的研究利用微载体技术规模化制备肠道病毒71型(Enterovirus 71,EV71)的方法。方法利用NBSCelliGen 310 5 L生物反应器进行Vero细胞微载体培养,考察了不同微载体Cytodex-1浓度(3、10、15、20 g/L)对Vero细胞生长代谢及细胞密度的影响,并且与细胞工厂(Cell factory,CF)中Vero细胞染毒后的病毒繁殖进行比较。分别采用上清液、洗涤液、洗脱液模式收毒,比较不同收毒方式中EV71的抗原含量及病毒滴度(CCID50)。结果采用10 g/L微载体浓度,批次培养方式培养Vero细胞120 h后,细胞密度可达5.47×106个/ml;当微载体浓度大于15 g/L时,由于葡萄糖消耗速度快,需采用灌注模式培养。按MOI 0.2染毒后,微载体培养的收毒时间比CF慢48 h,但其病毒滴度可达8.8 Log10CCID50/ml,约为CF的5倍。EV71与微载体存在离子交换吸附作用,按上清液加洗脱液方式收毒,抗原总量可达12 61 U/ml,约为CF的3倍。结论已成功建立了生物反应器微载体5 L发酵培养Vero细胞生产EV71的方法,为进一步EV71大规模培养以及疫苗研发奠定了基础。     

动物细胞发酵缶的混合与剪切性能评价

微载体悬浮培养动物细胞条件下,发酵缶的混合与剪切性能优劣对动物细胞的培养效果至关重要。本文研究了NBS动物细胞发酵缶的流体混合时间、搅拌功率和时均速度分布,探讨了微载体及动物细胞所受到的流体作用力的计算方法,为设计大规模动物细胞发酵缶不变提供了部分依据。     

生物反应器大规模培养脊髓灰质炎病毒

目的应用生物反应器大规模培养脊髓灰质炎病毒。方法用7L、75L和550L生物反应器逐级放大培养Vero细胞,每天取样进行细胞计数。在550L生物反应器中培养脊髓灰质炎病毒,观察细胞病变,并检测病毒感染性滴度。结果一级细胞培养(灌流培养法)、二级细胞培养(循环培养法)和三级细胞培养(批培养法)的平均细胞密度分别为2.79×106、2.38×106和1.04×106个/ml,一级培养的细胞倍增数最高;病毒培养体积达350L,病毒收获液滴度大于7.625lgCCID50∕ml。结论通过三级放大工艺,可大规模培养脊髓灰质炎病毒。     

生物过程工程研究在创新生物医药开发中应用的驱动力——生物反应器

近年创新生物医药在生物产业中的比重逐渐增大,也给生物产业带来了巨大的经济效益,靶点筛选及分子构建等上游技术的进步是促进生物医药进步的主要原因.随着目前细胞培养技术在生物医药生产中的广泛应用,对细胞培养技术的要求也不断提高,同时细胞培养技术的实现载体——生物反应器的技术改进和创新也越发凸显其重要性.本文介绍了生物反应器在创新生物医药产业中的应用种类、发展趋势及发展驱动力,回顾了全球范围内新型生物反应器的发展成果,包括新型反应器技术及过程分析技术在生物医药中的应用,最后,分析了中国生物反应器的发展现状与问题,指出了生物反应器的发展与进步应以提高生物培养过程的稳定性最终提高产品的质量而不是以提高产量为主要目标.本文详细阐述现代生物反应器技术及基于"质量源于设计"的质量控制理念在其中所起到的关键作用,以及生物反应器的技术发展的现状和未来走向.     

动物细胞培养生物反应器研究进展

介绍了搅拌式生物反应器、非搅拌式生物反应器、粘性泵生物的反应器、膜式旋转细胞培养器、旋转式细胞／组织三维培养器以及脉冲式生物反应器，其中有新型的，也有改进传统型的。     

Gas‐Liquid Mass Transfer Characteristics in a Rotating‐Drum Bioreactor

The oxygen volumetric mass transfer coefficient is a key parameter to characterize the performance of aerobic bioreactors. A novel rotating‐drum bioreactor (RDB) fitted with a sparger as proposed in a previous work has demonstrated its excellent gas‐liquid mass transfer performance. To provide primary information on the design and scale‐up of the novel RDB, effects of reactor configuration including the number and width of lifters and operation conditions such as rotational speed, aeration rate, and solid volume fraction on mass transfer performance were systematically investigated in a new medium‐sized RDB. Compared with the stirred bioreactor and traditional RDBs, this new RDB exhibits better mass transfer performance. Taking both operational and reactor configuration parameters into consideration, an empirical correlation to predict the volumetric mass transfer coefficient in this type of RDBs was proposed which is valuable for its design and scale‐up.     

Modeling of the Oxygen Transfer Characteristics of a ‘See‐Saw’ Bioreactor

Animal cell line culture is difficult in the existing conventional bioreactors. A substantial amount of animal cells are destroyed by the impinging fan blades or entrapment inside the bubbles. An endeavor has been made to design and develop a new type of bioreactor suitable for animal cell culture. The bioreactor is named a ‘see‐saw’ bioreactor from its underlying principle of operation. In this paper, the oxygen transfer characteristics of the ‘see‐saw’ bioreactor are modeled and tried to be verified.     

Assessment of Relevant Factors Influencing Lipolytic Enzyme Production by Thermus thermophilus HB27 in Laboratory‐Scale Bioreactors

Nowadays, there is not much information on the large‐scale production of thermostable lipolytic enzymes by thermophilic organisms. Therefore, in this study the lipolytic enzyme production by Thermus thermophilus HB27 in laboratory‐scale bioreactors was undertaken. In order to determine the most suitable bioreactor, two configurations were investigated: stirred‐tank and airlift bioreactor. It was demonstrated that the stirred‐tank configuration led to the highest lipolytic extracellular activities, about 2.3‐fold higher than those found in the corresponding cultivation in the airlift bioreactor. On the other hand, the influence of several factors such as culture nutrients concentration, aeration, and agitation rate on the production of thermophilic lipolytic enzymes in a 5‐L stirred‐tank bioreactor was assayed. At reduced nutrients concentration (50 % with respect to the basal medium), a higher product/biomass yield was attained, without any operational problems. From the relationship between mass transfer coefficient (K_La), aeration, and agitation rates it was concluded that there is a lesser dependence on the aeration than the agitation rate. In addition, the mechanical stirring of the bioreactor could tear the membranes that contain the rotund bodies often found in cultures of thermophilic microorganisms, thus increasing the extracellular enzyme production.     

Perfusable System Using Porous Collagen Gel Scaffold Actively Provides Fresh Culture Media to a Cultured 3D Tissue

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.     

Multiscale simulation of nutrient transport in hollow fibre membrane bioreactor for growing bone tissue: Sub-cellular scale and beyond

Recent experimental studies suggest that hollow fibre membrane bioreactors (HFMBs) may be used to grow 3-D bone tissues in the laboratory, which may then be implanted into patients to repair skeletal defects. The HFMBs mimic the capillary network that exists in bones and are very effective in supplying nutrients to cells (to maintain cell metabolism) and removing waste products (e.g., excreta from micro-organisms, etc.). In order to guide the design of HFMBs for bone tissue engineering, it is necessary to elucidate the quantitative relationships between the cell environment and tissue behaviour in HFMBs and their relationship with nutrient supply. However, the nutrient transport processes in these bioreactors depend on several scales: from the scale of the individual cell to the scale of the bioreactors (laboratory scale). Further, the significance of the mass transfer processes is different from one scale to another. At the sub-cellular scale (i.e., within individual cell), the transport processes are dominated by diffusive-reaction mechanisms. At the extracellular matrix, these processes are primarily diffusion dominated. The transport of nutrients in the capillary network is convection dominated. At the scale of the laboratory device, the transport behaviour is governed by non-linear coupled convection-diffusion and reaction processes. Therefore, to characterise the ‘overall’ nutrient transfer processes and function of the HFMB, one needs an understanding of the processes at the smaller scale (e.g., sub-cellular scale) and their manifestation at larger scale, such as the bioreactor.This paper presents an approach for modelling and simulating nutrient transport in HFMB for growing bone tissues where the separations of scales from individual cell to the scales of bioreactor are considered. We use direct numerical simulation (finite element method) instead of more tedious applied mathematics based upscaling theorems for modelling nutrient transport in HFMB. The advantage of this approach is that it does not rely on the determination of averaged transport properties (e.g., diffusion coefficient) that appear in averaged transport equations which are often difficult to measure experimentally or may not have significant physical meaning. In this paper, the developed computational framework is used to upscale the mass transfer processes at sub-cellular scale to the scale of HFMB (laboratory scale). The developed framework is then employed to carry out a systematic analysis of the influence of various process parameters of HFMB (e.g., fluid velocity, cell density, cellular size, etc.) on the nutrient transport behaviour. It is envisaged that the developed multiscale tool will provide better understanding of the functioning of HFMB for the purposes discussed above.     

Über den Zusammenhang von Fleisch- und Fettbildung bei wachsenden Tieren

Relationship Between the Formation of Meat and Fat in Growing Animals Production of slaughter animals as human food means, primarily, the production of protein and fat. The production of protein is by far the most important objective. In contrast, the animal fat is generally considered as an undesirable component of foods. Although the arguments against the use of animal fats compared to vegetable fats are only to some extent justified, efforts have been made since many years to reduce the fat content and increase the proportion of meat by various means, such as breeding, special feeding and other techniques for raising animals. However, considerable amounts of fat are still produced besides meat in the production of slaughter animals. In swine and cattle the extent of formation of meat and fat during various stages of growth, the distribution of fat in various parts of the animals, the differences in fatty acid composition and means to alter them are discussed.     

生物转化温室气体生产单细胞蛋白的研究进展

全球人口持续增长使得肉、蛋和乳制品等生活品的需求大幅增加,同时也对传统动物饲料的供应带来了空前的挑战。微生物能够利用二氧化碳（CO₂）、甲烷（CH₄）等多种原料合成高蛋白含量的单细胞蛋白（single cell protein,SCP）以用于饲料或食品加工。生物转化CO₂和CH₄制备SCP不但可以扩展蛋白生产渠道和缓解各方面对蛋白的需求,而且也有望降低其生产成本,实现节能减排。从SCP合成及生产现状出发,探讨了好氧性甲烷菌和微藻利用温室气体的代谢路径、生物转化工艺、生物反应器设计的研究进展和未来应用前景,同时结合研究数据对生物转化温室气体制备SCP的经济可行性进行了初步评价和比较。     

Verarbeitung tierischer Rohstoffe

Processing of Animal Raw Materials In the commercial production of fish and meat products which are intended for human consumption, waste products are obtained which can be used, e. g. in the feeds industry, for the production of glue and gelatine or in the chemical industry. Generally, during further processing of these waste materials, following a cell opening stage, they are separated into high-protein solids and the fat/oil and water phases. As an ecologically and economically advantageous alternative to the conventional methods in which the cells are generally opened by thermal treatment, as new method - the ELCRACK®-process, has been developed. The ELCRACK®-process is a combination of the product-saving opening of animal cells under the influence of a high-voltage electrical field at comparatively low temperatures and the subsequent mechanical solid-liquid separation which takes place in a screw press and the centrifuges. Taking bone processing as an example, this process is explained more in detail and compared with conventional methods.