红豆杉悬浮细胞放大培养的细胞生长与紫杉醇合成动力学

研究了在Murashige&skoog s(MS)和 6 2号两种不同的培养基中 ,红豆杉细胞悬浮细胞从摇瓶到 1 0L机械通气搅拌式反应器放大培养过程中细胞生长与紫杉醇合成动力学 .结果表明 :尽管在不同的培养条件下 ,细胞生长曲线均呈现“S”型 .紫杉醇在延迟期与指数生长期中基本上没有积累 ,而且随着培养规模的增大 ,紫杉醇的含量逐渐降低 .进一步对各级放大培养的细胞生长 ,比生长率与胞内外紫杉醇合成量进行分析 ,发现MS利于细胞生长但不利于紫杉醇合成 ,而 6 2号则相反 .根据此文的结果 ,提出了红豆杉细胞培养条件的优化和大规模细胞培养生产紫杉醇应采取的策略     

红豆杉细胞两相培养生产紫杉醇的研究

研究了两相培养系统中红豆杉细胞的生长与代谢规律,经４０天培养,细胞生物量为１７．８５ｇ／Ｌ（干重）,紫杉醇产量为３０．１９ｍｇ／Ｌ。     

条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产的影响

在改良的B5培养基中加入不同浓度的聚乙二醇对东北红豆杉培养细胞进行摇瓶培养,通过不同时期取样并测定细胞鲜,干重及用HPLC测定紫杉醇的含量,发现聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产均有明显的促进作用,聚乙二醇为10g/L时,对细胞生长最为有利,细胞培养16d可达到最大生物量,其平均鲜重为28．73g/瓶,增重3．8倍,平均干重为2．14g/瓶,增重3．1倍,聚乙二醇为20g/L,对紫杉醇的生产最有利;细胞培养25d时,培养基中紫杉醇的含量达到最高水平,其含量为2350ug/L,是不加聚乙二醇的11倍。     

红豆杉细胞悬浮培养结构化数学模型的探讨

用１０Ｌ机械搅拌式生物反应器悬浮培养红豆杉细胞,得到细胞生长、基质消耗和紫杉醇合成动力学曲线。经过代谢动力学分析建立了结构化数学模型。并将模型值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

超声波对红豆杉悬浮细胞生长及紫杉醇释放的研究

分析了超声波对中国红豆杉悬浮细胞培养的生长,紫杉醇合成及释放的影响,细胞对不同强度及作用时间的超声波反应不同,用38kHz,120s的超声强度处理悬浮细胞,紫杉醇胞外释放率由对照的10％左右提高到40－50％,总产量提高了47％,超声波处理植物细胞,提供了在保持细胞生长的前提下有效刺激胞内次生代谢物的简易操作方法。     

前体物对红豆杉培养细胞中紫杉醇生物合成的影响

本文报道了添加７种紫杉醇前体物／调节物后,红豆杉（Ｔ．ｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇｅｒ）Ｒｅｈｄ）ＴＣ１５８细胞系的反应,在红豆杉细胞悬浮培养２５天时,分别加入不同浓度乙酸钠,苯甲酸钠,Ｌ－苯丙氨酸,甘氨酸,丝氨酸、α－蒎烯,松节油。试验结果表明,各前体物对红豆杉细胞生长无明显影响,均不同程度地促进了紫杉醇的合成。     

云南红豆杉细胞发酵培养的研究

利用云南红豆杉(Taxus yunnanensis)细胞悬浮培养的最佳培养条件,进行了细胞10L规模的发酵培养研究。对细胞发酵培养过程中的pH值变化、糖利用率以及细胞生长周期等实验参数进行了测定。已进行的3次发酵培养的实验结果表明:培养细胞的生长率已达0.4g/L.d(即12.0g/L),培养细胞中紫杉酵的含量为0.119％(其中培养液中的紫杉醇含量约占42％)。初步建立了优化的云南红豆杉细胞大量培养系统。     

九连小蘖悬浮培养细胞生理生化研究 Ⅰ.细胞生长与培养液的电导率和过氧化物酶活性的变化

本文报道了九连小蘖细胞悬浮培养过程中,细胞生长与培养液的电导率、pH值、可溶性糖含量及过氨化物酶活性的变化。实验表明细胞生长曲线与培养液的电导率、可溶性糖含量变化的曲线恰成镜像关系。而细胞生长曲线与培养液的过氨化物酶活性变化的曲线相互平行。从而,可以通过监测培养液的电导率和过氧化物酶活性的变化来了解细胞生长状况,并可作为植物细胞培养过程中生物量增长的参考指标。     

九连小蘖悬浮培养细胞生理生化研究 Ⅰ.细胞生长与培养液的电导率和过氧化物酶活性的变化

本文报道了九连小蘖细胞悬浮培养过程中,细胞生长与培养液的电导率、pH值、可溶性糖含量及过氨化物酶活性的变化。实验表明细胞生长曲线与培养液的电导率、可溶性糖含量变化的曲线恰成镜像关系。而细胞生长曲线与培养液的过氨化物酶活性变化的曲线相互平行。从而,可以通过监测培养液的电导率和过氧化物酶活性的变化来了解细胞生长状况,并可作为植物细胞培养过程中生物量增长的参考指标。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

Determination of Mammalian Cell Counts,Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls^1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques⁶.Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.     

Transformations of the Cell Center during Cell Differentiation

A question was posed as to how the multicomponent and polyfunctional organelle dynamically changes during metazoan ontogenesis. The centrosome structure is gradually formed and its functions are switched on during early embryogenesis, one of which is the cell center formation. During cell differentiation, the condition of the cell center and surrounding structures may be different: first, the cell center is quite distinct; second, the cell center is absent due to redistribution of the microtubule organizing centers; third, the cell center disappears due to reversible or irreversible inactivation of the centrosome and other centers of microtubule organization. The assembly of the Golgi complex does not depend directly to the cell center presence. In some cell types, the Golgi complex is topologically associated with the cell center, while in others it exists as individual dictyosomes despite the cell center presence. In some other cell types, the common Golgi complex is assembled without the cell center, but in the presence of microtubules that are formed by noncentrosome centers of microtubule organization. In still others, degradation of both the cell center and the common Golgi complex takes place in the case of centrosome inactivation.     

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed¹ who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate².To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry¹. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments¹.The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection³ in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.     

Cell Physician: Reading Cell Motion

Cell motility is an essential phenomenon in almost all living organisms. It is natural to think that behavioral or shape changes of a cell bear information about the underlying mechanisms that generate these changes. Reading cell motion, namely, understanding the underlying biophysical and mechanochemical processes, is of paramount importance. The mathematical model developed in this paper determines some physical features and material properties of the cells locally through analysis of live cell image sequences and uses this information to make further inferences about the molecular structures, dynamics, and processes within the cells, such as the actin network, microdomains, chemotaxis, adhesion, and retrograde flow. The generality of the principals used in formation of the model ensures its wide applicability to different phenomena at various levels. Based on the model outcomes, we hypothesize a novel biological model for collective biomechanical and molecular mechanism of cell motion.