Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Expression pattern and polymorphism of three microsatellite markers in the porcine CA3 gene

Carbonic anhydrase III (CA3) is an abundant muscle protein characteristic of adult type-1, slow-twitch, muscle fibres. In order to further understand the functions of the porcine CA3 protein in muscle, the temporal and spatial distributions of its gene product were analysed and the association between the presence of specific polymorphisms and carcass traits in the pig was also examined. Real-time PCR revealed that the CA3 mRNA expression showed no differences with age in skeletal muscles from Yorkshire pigs at postnatal day-1, month-2, and month-4. We provide the first evidence that CA3 is differentially expressed in the skeletal muscle of Yorkshire and Meishan pig breeds. In addition, the whole pig genomic DNA sequence of CA3 was investigated and shown to contain seven exons and six introns. Comparative sequencing of the gene from three pig breeds revealed the existence of microsatellite SJ160 in intron 5 and microsatellite SJ158 and a novel microsatellite marker that includes a tandem repeat of (TC)_n in intron 4. We also determined the allele number and frequencies of the three loci in seven pig breeds and found that they are low polymorphic microsatellite markers. Statistical analysis showed that the CA3 microsatellite polymorphism was associated with dressing percentage, internal fat rate, carcass length, rib number and backfat thickness in the pig.     

Process development for functional membrane receptor production in mammalian cells

Two model G-protein coupled membrane receptors (GPCRs), aserotonin (5HT) and a metabotropic glutamate (mGlu) receptor, stablyexpressed in CHO cells were used to characterize cultureconditions for maximum receptor expression and functionalactivity in membrane preparations. Expression levels of the5HT receptor were affected by the growth phase of the cellculture. Maximum receptor density, as measured by ligandbinding per mg membrane protein, was observed when cells wereharvested in late exponential growth phase. Expression couldbe increased further by addition of 10 mM sodium butyrate andincubation at 31 °C for 24 hours prior to cellharvest. In contrast, functional activity as determined byagonist-stimulated GTPS binding was independent of the growthrate. For both receptors, butyrate treatment at decreasedtemperature negatively affected functional activity. The mGlureceptor membranes lost functional activity considerably whenthe cells were cultured in an agitated system either onmicrocarriers or as aggregates in suspension. Functionalactivity could be restored and further improved compared to acontrol grown in T-flasks when the cell culture was incubatedat 31 °C for 48 hours following a complete mediumexchange and omission of sodium butyrate.     

Automation of cell line development

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 μM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 μM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 μM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process. A. Salmén and K. Lindgren contributed equally to the work.     

苏南典型城镇耕地景观动态变化及其影响因素

基于高分辨率遥感影像获得3期土地利用数据,引入景观动态度和缩减强度指数分析了辛庄镇各村水田景观减少的速度和强度;利用转移矩阵和GIS地图叠加方法分析了辛庄镇水田景观变化的时空特征;应用冗余分析方法,直观地展现了主要的水田转变方式与各影响因子的定量关系。结果表明:1991-2009年间,研究区水田景观面积比例减少了30.5%,且呈加速下降趋势;各村水田景观缩减的规模、速度和强度差异明显;减少的水田主要被工业用地、鱼塘和居住用地占用,且这种现象集中发生在主要道路与河流周围。社会经济因子和区位因子对水田变化的空间差异具有很好的解释作用,且各因子对解释不同时段水田变化的空间分异的贡献率差异显著。     

小白撑根部二萜生物碱研究

从小白撑(Aconitum nagarum var.heterotrichum)根部分离出5个二萜生物碱,其结构通过光谱分析和化学方法鉴定为光翠雀碱(1),宋果灵(2),乌头碱(3),去氧乌头碱(4),和滇乌碱(5)。     

Distinct distribution patterns of prokaryotes between sediment and water in the Yellow River estuary

Applied Microbiology and Biotechnology - There are close exchanges between sediment and water in estuaries; however, the patterns of prokaryotic community assembly in these two habitat types are...