三种细胞培养模型的效果评价

原代大鼠肝细胞经不同体外细胞培养模型培养后 ,对肝细胞的酶渗出量、白蛋白分泌水平和细胞色素P450 1A(CYP 1A)活性进行了分析。结果显示 :在三种培养模型中 ,培养液的LDH水平随培养时间的延长而逐渐降低 ,但在单层培养 (MC)的第 5天 ,LDH水平明显升高 ,而夹层培养 (SC)的LDH在第 8天后没有检出 ,AST和ALT水平在整个培养过程中无明显变化。在生物反应器中 (bioreactor) ,肝细胞基础CYP1A活性可维持 2周以上 ,培养液中白蛋白的含量以生物反应器最高 ,其次是SC和MC。随着培养时间的延长 ,MC和SC中肝细胞CYP 1A活性逐渐降低 ,且MC肝细胞CYP 1A活性的下降速度比SC快。这种现象可被细胞色素P450 (P450 )的诱导剂如奥美拉唑和 3 甲基胆蒽 (3 MC)部分改变 ,MC肝细胞的诱导作用总是比SC肝细胞强。结果证实每一个体外细胞培养模型都有各自的优缺点 ,不同的体外细胞培养模型可以解决问题的不同方面     

波浪式生物反应器pH控制中PID控制算法的研究

目的:设计一种快速高效的波浪式生物反应器pH控制方法,缩短pH调节时间,并降低调节过程的系统超调。方法:根据pH调节特性,设计一种分段式变增益PID(proportion-integration-differentiation)控制算法,并通过仿真实验与传统PID控制进行对比,验证算法的有效性。结合波浪式生物反应器细胞培养的实际情况对该算法进行改进,得到四区段变增益PID控制算法,通过实际实验验证改进算法的有效性。结果:仿真实验表明分段式变增益PID控制相较于传统PID控制调节速度快且系统超调小;实际实验表明四区段变增益PID控制能迅速将pH调节到设定范围内,且稳定性好、精确度高。结论:四区段变增益PID控制能够对pH控制起到很好的调节作用,满足波浪式生物反应器细胞培养过程中对pH控制的要求。     

血管生物反应器中的血流动力学及其应用

血管生物反应器是血管组织体外培养过程中血管重建的关键设备,为血管组织的体外培养提供稳定的、与生物体内相似的体外环境。目前许多血管生物反应器在组织工程血管重建中得到广泛应用。血流动力学是新近引入血管生物反应器的重要研究理论。分析血流动力学在血管重建中的作用,探讨血管生物反应器中力学环境的相关问题及其在不同血管重建中的具体应用。     

一种用于骨组织工程生物反应器的设计

根据目前骨组织工程化培养生物反应装置的发展,结合骨组织的力学感知传导机理,研制了一种用于骨组织工程化培养和研究的生物反应器,其中,采用直接施加动态压缩载荷和灌流系统改善培养物的力学激励和传质过程.该反应器可为骨三维支架(包括有一定强度的支架)提供生理范围内不同大小、频率的压缩应变及其内表面不同的流体剪应力.这种生物反应器不仅为培养物形成了类似在体骨组织的力学激励,而且可能提供培养物营养和废物的有效运输.     

超宽带电磁波对离体淋巴细胞损伤效应初探

目的：由于电磁脉冲技术的广泛应用，暴露于电磁场中的人类健康可能受到影响，但一些特殊电磁波的生物效应，尤其是超宽带（ｕｌｔｒａ－ｗｉｄｅｂａｎｄＵＷＢ）电磁波生物效应研究较少，本实验应用培养淋巴细胞为模型，探讨其对细胞的损伤效应。方法：培养的淋巴细胞暴露于ＵＷＢ、峰值功率密度为１００Ｗ．ｃｍ－２，测定不同时间电磁波辐照后细胞死亡率和培养上清液中乳酸脱氢酶（ＬＤＨ）浓度变化。结果：ＵＷＢ电磁波辐照后细胞死亡率明显增高，培养上清液中ＬＤＨ浓度比正常对照显著增加，但两指标均不随培养时间变化而发生明显改变。结论：较低平均功率密度的ＵＷＢ电磁波能造成细胞损伤     

120例不同孕周羊水细胞培养结局及影响因素初探

目的探讨不同孕周羊水细胞培养的结局及相关影响因素。方法对120例不同孕周引产孕妇在B超引导下行羊膜腔穿刺术,取羊水细胞培养;统计不同孕周羊水细胞培养的成功率,并对不同孕周孕妇的年龄、外周血WBC、羊水WBC指标与培养结局进行相关分析。结果 120例羊水细胞培养成功114例,培养成功率为95.0%;18周到22周羊水细胞培养成功率最高,为100%;孕妇的年龄是不同孕周下影响羊水细胞培养成功率相关因素(P<0.05)。结论 18周到22周进行羊水细胞培养能有效提高羊水细胞培养的成功率;不同孕周条件下孕妇的年龄对羊水细胞培养结局有影响。     

中国疫苗生产技术的领跑者

<正>引进·消化·吸收·创新国际领先技术打造优秀民族企业辽宁成大生物股份有限公司于2002年6月在沈阳市成立,是由辽宁成人股份有限公司(G600739)和辽宁省生物医学工程研究院共同组建的高新技术企业。辽宁成大生物股份有限公司于国内率先引进世界先进的生物反应器高密度微载体Vero细胞培养技术和四柱层析系统,建立了大规模生产病毒类疫苗的技术平台。人用狂犬病疫苗年生产能力达到800万人份,成为全球最大的供应商之一。     

微载体技术高密度培养MRC-5细胞初探

目的 通过探索MRC-5细胞的微载体培养条件,以期建立MRC-5细胞的高密度培养方法.方法 用3种不同培养基（MEM、M199、DMEM）分别培养3.0×104/mL的MRC-5细胞,观察它们的细胞增殖及传代能力;在Spinner培养系统中,用1.0 g/L Cytopore 2和3.0 g/L Cytodex 3分别培养密度为3.70× 105/mL和2.98×105/mL的MRC-5细胞,观察两种载体对细胞生长代谢和细胞密度的影响,筛选出最适宜的培养基及微载体.使用5 L CelliGen310生物反应器对筛选获得的培养基及微载体进行MRC-5细胞的灌流培养初步摸索.结果 MRC-5细胞在MEM、M199、DMEM培养基中培养96 h细胞分别增殖了7.0、4.4和3.0倍;在MEM培养基中连续传代至5次以上,细胞增殖稳定,1：3传代72 h形成致密单层,而在M199和DMEM培养基中连续传代均未能获得理想的细胞形态.经96～120 h的培养后,使用1.0 g/L Cytopore 2培养的MRC-5细胞密度达到2.20× 106/mL高于使用3.0 g/L Cytodex 3的1.12×106/mL,且前者葡萄糖和乳酸代谢较后者更为活跃.首选MEM和Cytopore 2作为MRC-5细胞的灌流培养体系.在5L生物反应器中以1.0 g/L Cytopore 2和2.5LMEM培养基培养MRC-5细胞至144h,细胞密度达到1.54×106/mL.结论 初步建立的MEM Cytopore 2微载体细胞培养方法能够获得高密度、活性良好的MRC-5传代细胞.     

运行参数对厨余垃圾生物水解反应影响的中试研究

随着垃圾分类在全国的实施，产生了巨量厨余垃圾，需要建立新的终端设施以对其进行资源化利用。机械生物消融（EMBT）技术是一种可将垃圾中有机资源最大化利用的技术，生物水解反应器是EMBT技术的核心单元，可以将不同类型有机质从固相高效转化为液相，通过挤压后得到的有机浆液可应用于高效湿式厌氧反应器。经水解酸化后的浆液，产气率更高，可缩短厌氧反应器停留时间并提高其稳定性。通过开展中试试验，对比不同的停留时间、搅拌速率和温度条件下厨余垃圾减量化率和浆液COD等参数的变化规律，并与直接挤压参数进行对比，发现最佳结果是在2 d停留时间下，厨余垃圾减量化率可达64%,COD转化总量达到247 kg/t以上，生物水解后产生浆液的VS产气率可达877.6 mL/g，该结果优于厨余垃圾直接挤压对应的减量化率以及产生浆液的各项参数。     

基于罗叶泵的搏动式组织工程培养体系的构建

目的:设计并制作基于心室辅助泵-罗叶泵的搏动式组织工程培养体系,并完成其基本性能测试。方法:根据血管组织工程的需求进行生物反应器的设计,生物反应器主体部分由玻璃构成。启动罗叶泵,调节罗叶泵的输出压力和频率,利用心电监护仪监测生物反应器内流体压力的大小和频率,利用流量计测量其实际流量。结果:生物反应器的主体部分由玻璃制备而成。所组装的培养系统经14d运行无松脱、无渗漏。心电监护仪显示罗叶泵产生的压力波形为脉冲波。当罗叶泵的输出频率固定时,循环管路中的实测压力和流量随罗叶泵输出压力的增加而增加。当罗叶泵的输出压力固定时,循环管路的实测压力和流量随输出频率的增大而增加。结论:本研究所开发的组织工程培养体系稳定性好,可产生脉动刺激,可初步应用于血管组织工程三维培养。通过调节输出压力、频率等参数,可产生不同的流体规律,可用于调节心血管组织工程的微环境。     

A scale-down model of 4000-L cell culture process for inactivated foot-and-mouth disease vaccine production

The anticipated increasing demand for inactivated foot-and-mouth (FMD) disease vaccine calls for its larger production capacity, while development of a large-scale process typically requires high running cost and has very limited experimental throughput at manufacturing scale. Thus, an economic scale-down model of representing a large-scale process becomes necessary and essential. In this study, we used a systematic approach to establish a scale-down model representing a 4000-L culture process for FMD vaccine production by suspension BHK-21 cells. In detail, we firstly compared hydrodynamic properties of three bioreactors (14-L, 800-L and 4000-L) under three different conditions (equivalent mixing time, equivalent shear stress and equivalent volumetric power). We figured out equivalent volumetric power (P/V) potentially as an appropriate scale-down strategy, since it resulted in comparable calculated hydrodynamic parameters among three bioreactors. Next, we used computational fluid dynamics (CFD) simulation to provide more details about hydrodynamic environments inside the bioreactors, which supports the reliability of this scale-down strategy. Finally, we compared cell growth, metabolites, vaccine productivity and product quality attributes during FMD vaccine production by BHK-21 cells and observed very close performances among three bioreactors, which once again demonstrates the robustness of this scale-down model. This scale-down strategy can be applied to study variations and critical quality attributes (CQAs) in the resultant production process based on quality by design (QbD) principles, aiming at further more efficient optimization of vaccine production.     

自然流产胚胎绒毛细胞培养方法比较

目的:探讨自然流产胚胎绒毛细胞培养的简单有效方法。方法:取57例早期自然流产胚胎绒毛组织分别采用改良组织块培养法和酶消化法进行培养。结果:改良组织块培养法培养成功51例(89.5%),酶消化法培养成功42例(73.7%),两者比较差异有统计学意义(P<0.05)。结论:改良组织块培养法用于自然流产胚胎染色体诊断简单可行,成功率高。     

Evaluation of four cell lines for assay of infectious adenoviruses in water samples

Human viral contamination in drinking and recreational waters poses health risks. The application of PCR-based molecular technology has advanced our knowledge of the occurrence and prevalence of human viruses in water; however, it has provided no information on viral viability and infectivity. Four human cell lines were compared for their sensitivity to different serotypes of human adenoviruses using the TCID₅₀ test. The sensitivity of each cell line varied with different serotypes of adenovirus. Human embryonic kidney cell line 293A and human lung carcinoma cell line A549 were the most sensitive, especially to enteric adenovirus 40 and 41. Plaque assay of primary sewage samples showed 293A can detect viral plaques in 7 of 13 primary sewage samples tested. Adenoviruses were also isolated using 293A from environmental water concentrates. Cloning and sequencing of environmental adenoviral isolates indentified them to be aligned with adenoviruses serotype 40 and serotype 5. The result of this study suggests that plaque assay with 293A cell line is suitable for detection of adenovirus in the aquatic environment. Combining this cell culture with molecular methods for viral assay in the aquatic environment will provide critical information for risk assessment.     

An evaluation of second generation tissue culture rabies vaccines for use in man: a four-vaccine comparative immunogenicity study using a pre-exposure vaccination schedule and an abbreviated 2-1-1 postexposure schedule

In a double-blind comparative trial the immunogenicity of three new tissue culture rabies vaccines was evaluated, using a commercial human diploid cell vaccine (HDCV) lot as the reference. Two different vaccination regimens, a pre-exposure schedule, and an abbreviated 2-1-1 postexposure schedule (two doses of the vaccine applied bilaterally on day 0, with subsequent single doses given on days 7 and 21) were employed. In both, two of the new vaccines, purified chick embryo cell vaccine and purified Vero rabies vaccine, induced an antibody response equivalent to that of HDCV, while the geometric mean titres for the fetal bovine kidney cell vaccine were somewhat lower. The 2-1-1 regimen, a candidate regimen for economical rabies postexposure treatment, evoked a rapid and high titre antibody response with all four vaccines, peaking on day 14.     

In vitro cell culture infectivity assay for human noroviruses

Human noroviruses cause severe, self-limiting gastroenteritis that typically lasts 24-48 hours. Because of the lack of suitable tissue culture or animal models, the true nature of norovirus pathogenesis remains unknown. We show, for the first time, that noroviruses can infect and replicate in a physiologically relevant 3-dimensional (3-D), organoid model of human small intestinal epithelium. This level of cellular differentiation was achieved by growing the cells on porous collagen-I coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in situ hybridization provided evidence of norovirus infection. Cytopathic effect and norovirus RNA were detected at each of the 5 cell passages for genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts that used differentiated monolayer cultures failed.     

Vaccine against infectious bovine keratoconjunctivitis: a new approach to optimize the production of highly piliated Moraxella bovis cells

Pili are the principal antigens and virulence factors of Moraxella bovis, the etiological agent of infectious bovine keratoconjunctivitis (IBK). Although it has been reported that the low efficacy of whole cell vaccines against IBK is mainly due to the difficulties in keeping the cellular piliation level of M. bovis during the growth of bacteria in stirred bioreactors, the problem has not yet been overcome because the mechanisms involved in the loss of piliation are still not fully clarified. In this work we found that during the culture of M. bovis in liquid media, around 15% of the cells changed from piliated to non-piliated phenotypes at the end of the growth. Nevertheless, we demonstrated that the main cause of cellular piliation loss in M. bovis growing in stirred and/or sparged bioreactors is due to shear forces, which are a function of the volumetric gassed power drawn (P(g)V(-1)). Therefore, we tested here the use of bubble column bioreactors to protect M. bovis cell-bound pili from mechanical agitation damage effects. These bioreactors operated at a superficial air velocity of 0.0065 m s(-1) yielded a cellular piliation level of 25%, in contrast to 1% obtained for stirred bioreactors. The addition of carboxymethylcellulose (CMC) at 0.10% (w v(-1)) to culture medium proved to be suitable to improve the final piliation level (65%). We demonstrated by FT-IR spectroscopy and ELISA technique, that this chemical additive has a pili protective role interacting with the cells but without affecting pili antigenic properties.     

影响重组CHO C28细胞HBsAg表达的重要因素

摸索乙肝疫苗生产过程中细胞培养的最佳方案。探究重组CHO细胞培养过程中不同厂家的血清、DMEM培养基等相关原材料对HBsAg表达及细胞生长形态的影响。     

Infection dynamics and virus-induced apoptosis in cell culture-based influenza vaccine production—Flow cytometry and mathematical modeling

Cell culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus–host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale microcarrier culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.     

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens’ eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms.