高通量微型生物反应器的研究进展

近年来,哺乳动物细胞培养技术发展迅猛,基于此技术的生物制药行业更是异军突起。在激烈的生物药市场竞争中,缩短研发时间和降低研发成本是制胜的关键。与传统的生物反应器相比,高通量微型生物反应器具有操作简单、运行通量高、实验重复性好等优点,可大大缩短研发周期,降低人力、物力成本,因此成为了生物制药行业最新的研究热点之一。目前,已成功应用于生物药物研发的微型生物反应器有Simcell ^TM、Ambr 15 ^TM、Ambr 250 ^TM等,分别适用于工艺开发中的不同阶段。以上述三种微型生物反应器为例,介绍高通量微型反应器在哺乳动物细胞培养工艺开发中的研究现状及发展前景。     

生物反应器及其研究技术进展

阐述了生物反应器设计、放大的新理念及关键技术发展,并在此基础上综述了应用于生物技术产品生产的生物反应器的主要发展趋势,包括以代谢流分析为核心的生物反应器系统、基于计算流体力学模拟技术的传统发酵罐改良、微型生物反应器、动物细胞反应器和酶反应器。     

国产生物反应器大规模高密度培养Vero细胞

本文考察了在2．5LcelliGen细胞培养器和国产20LcellCul-20细胞培养生物反应器中采用微载体技术培养细胞的情况。分析了用cellcul-20细胞培养生物反应器进行大规模培养时细胞的生长、代谢规律,研究了从2．5L扩大到20L规模的细胞转移条件。采用微载体球间直接转移技术。提高了接种效率,减少了接种步骤和污染机会。当国产GT一25微载体用量为5g／L,采用连续灌注工艺培养vero细胞,在国产20L cellCul—20细胞培养生物反应器中,连续培养5天,细胞数增加7倍,细胞密度超过1．0×10⁷ cells／m】。本文开发的细胞培养工艺,对于中试及工业规模的动物细胞大量培养具有一定的指导意义。     

植物组织培养生物反应器技术研究进展

从植物大规模组织培养的特点、反应器类型和反应器中微环境等方面介绍了生物反应器技术在药用植物微繁殖和天然产物细胞生产中的研究进展。     

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。     

VERO细胞生物反应器放大培养初探

目的:研究用生物反应器放大进行Vero细胞微载体培养,实现生物反应器之间Veto细胞放大培养.方法:5L微载体生物反应器以10g/L微载体浓度培养Vero细胞,96h时经漂洗、消化、接种于30L微载体生物反应器,实现放大后的30L微载体生物反应器细胞怏速增殖,期间对不同时期的微载体细胞进行细胞计数、细胞代谢分析和形态观察.结果:5L生物反应器细胞经过96h灌注培养,平均细胞密度达到7.81×10~6cells/mL.5L微载体细胞放大到30L微载体生物反应器,平均细胞收获率为32.3%;放大到30L生物反应器后经过144h培养,细胞密度达到9.19×10~6cells/mL;放大后的细胞代谢途径依然以葡萄糖氧化代谢乳酸为主.结论:生物反应器由5L到30L进行Veto细胞放大培养是可行的.     

发展我国大规模细胞培养生物反应器装备制造业

生物反应器在生物工程装备制造业的产业化方面具有极其重要的意义。在分析了生物反应器产业的产品特点后,对我国生物反应器产业的现状和今后发展作了讨论。对以代谢流分析为核心的生物反应器、带pH测量与补料控制的摇床、动物细胞大规模培养生物反应器研制、生物反应器中试系统设计、大型生物反应器设计与制造技术研究等几个重要开发内容进行深入讨论,最后对细胞过程的系统生物学研究与生物反应器作了展望。     

封闭式光生物反应器研究进展

国际上80～90年代,封闭式光生物反应器是微藻生物技术的重要研究热点,也是微藻生物技术产业化的关键技术之一。本文较全面地介绍了用于微藻大规模培养的封闭式光生物反应器研究现状。将封闭式光生物反应器分为柱式、管式、板式和光导纤维反应器等类型。工业放大前景的管式和板式光生物反应器采取了典型个案分析的方法,列表比较了典型反应器的主要技术参数,并对它们的技术发展趋势进行了归纳总结。     

微载体灌注培养制备Vero细胞狂犬病疫苗

本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术。在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6~7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0~8.5logLD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到《中国生物制品规程》2000年版要求。实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行。     

转基因动物生物反应器研究及产业化进程

<正>概述了生物反应器的概念与分类,介绍并比较了不同动物生物反应器的研究现状和发展前景,提出乳腺是生产结构和功能复杂的重组蛋白的最佳生物反应器,具有广泛的应用潜力,可在生产新型牛奶和药用珍稀蛋白等方面为人类做出巨大的贡献。同时指出了包括乳腺反应器在内的动物生物反应器存在的问题,对乳腺生物反应器的应用前景进行了展望。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Vascular volumes and hematology in male and female runners and cyclists

To examine the hypothesis that foot-strike hemolysis alters vascular volumes and selected hematological properties is trained athletes, we have measured total blood volume (TBV), red cell volume (RCV) and plasma volume (PV) in cyclists (n = 21) and runners (n = 17) and compared them to those of untrained controls (n = 20). TBV (ml x kg(-1)) was calculated as the sum of RCV (ml x kg(-1)) and PV (ml x kg(-1)) obtained using 51Cr and 125I-labelled albumin, respectively. Hematological assessment was carried out using a Coulter counter. Peak aerobic power (VO2peak) was measured during progressive exercise to fatigue using both cycle and treadmill ergometry. RCV was 15% higher (P < 0.05) in male cyclists [35.4 (1.0), mean (SE); n = 12] and runners [35.3 (0.98); n = 9] compared to the controls [30.7 (0.92); n = 12]. Similar differences existed between the female cyclists [28.2 (2.1); n = 9] and runners [28.4 (1.0); n = 8] compared to the untrained controls [24.9 (1.4); n = 8]. For the male athletes, PV was between 19% (cyclists) and 28% (runners) higher (P < 0.05) in the trained athletes compared to the untrained controls. The differences in PV between the female groups were not significant. Although the males had a higher (P < 0.05) TBV, RCV and PV than the females, no differences between cyclists and runners were found for either gender. Mean cell volume was not different between the athletic groups. VO2peak (ml x kg(-1) x min(-1)) was higher (P < 0.05) in both male [68.4 (1.5)] and female [54.8 (2.1)] runners when compared to the untrained males [47.1 (1.0)] and females [40.5 (2.1)]. Although differences existed between the genders in VO2peak for both cyclists and runners, no differences were found between the athletic groups within a gender. Since the vascular volumes were not different between cyclists and runners for either the males or females, foot-strike hemolysis would not appear to have an effect on that parameter. The significant correlations (P < 0.05) found between VO2peak and RCV (r = 0.64 and 0.64) and TBV (r = 0.82 and 0.63) for the males and females, respectively, suggests a role for the vascular system in realizing a high aerobic power.     

Microbial and bioconversion production of D-xylitol and its detection and application

D-Xylitol is found in low content as a natural constituent of many fruits and vegetables. It is a five-carbon sugar polyol and has been used as a food additive and sweetening agent to replace sucrose, especially for non-insulin dependent diabetics. It has multiple beneficial health effects, such as the prevention of dental caries, and acute otitis media. In industry, it has been produced by chemical reduction of D-xylose mainly from photosynthetic biomass hydrolysates. As an alternative method of chemical reduction, biosynthesis of D-xylitol has been focused on the metabolically engineered Saccharomyces cerevisiae and Candida strains. In order to detect D-xylitol in the production processes, several detection methods have been established, such as gas chromatography (GC)-based methods, high performance liquid chromatography (HPLC)-based methods, LC-MS methods, and capillary electrophoresis methods (CE). The advantages and disadvantages of these methods are compared in this review.