不同冷冻速率冻存C6/36细胞复苏效果比较

[目的]选择C6/36细胞最适宜冷冻速率。[方法]用4种冷冻速率冻存处于对数生长期的C6/36细胞,运用MTT法测定细胞活性,计算冷冻存活率,并同步观察复苏细胞形态。[结果]A法冷冻存活率(92.9%±1.5%)高于B法、C法和D法(71.6%±1.8%、69.9%±2.1%和35.2%±1.2%)。光镜观察,A法冻存后细胞,复苏即时镜下90%以上的细胞呈现透明,类圆形饱满良好的活细胞形态;而B法、C法和D法冻存的细胞都有不同程度的形态性破坏,可见细胞碎片。[结论]A法是C6/36细胞的最适宜冷冻速率,冷冻速率的改变对其冷冻存活率的影响很大(P<0.01)。     

生殖医学（辅助生殖技术）——精子快速冷冻时细胞损伤机制的研究：细胞内无冰晶形成

物理应激在冷冻保存的细胞应答中有着重要的作用。随着温度降低，细胞外液浓缩，精子细胞长期在高浓度环境中可能导致细胞高渗性脱水。因此，细胞损伤可能是因为解冻时渗透压失衡造成的。研究旨在观察保存在甘油中或等渗液中的精子，以不同的速率冷冻，然后用冷冻扫描电镜和冷冻置换技术观察其超微结构，判断是否有细胞内冰晶形成，或者存在另外的细胞损伤机制。     

胚胎玻璃化冷冻研究进展

玻璃化冷冻胚胎具有简便、迅速、经济、避免冰晶形成等优点，受到越来越广泛的关注。大量实验研究及初步临床应用结果表明，应用玻璃化冷冻技术能够保存多个种属不同发育阶段的胚胎。但是，目前玻璃化冷冻胚胎尚不成熟，应用合理的玻璃化冷冻溶液以及提高玻璃化冷冻速率等均是影响玻璃化冷冻胚胎的重要因素。     

精子快速冷冻时细胞损伤机制的研究:细胞内无冰晶形成

物理应激在冷冻保存的细胞应答中有着重要的作用。随着温度降低,细胞外液浓缩,精子细胞长期在高浓度环境中可能导致细胞高渗性脱水。因此,细胞损伤可能是因为解冻时渗透压失衡造成的。研究旨在观察保存在甘油中或等渗液中的精子,以不同的速率冷冻,然后用冷冻扫描电镜和冷冻置换技术观察其超微结构,判断是否有细胞内冰晶形成,或者存在另外的细胞损伤机制。     

临床低温外科装置

低温冷冻在临床上可用来杀死各类肿瘤组织，冷冻治疗具有许多优越性，如手术过程出血少、痛苦小。本文介绍低温外科装置的工作过程及装置结构。最后根据临床应用对装置的工作压力、冷冻速率、复温速率等重要参数进行讨论。     

卵丘细胞在未成熟卵母细胞玻璃化冷冻保存中的作用

卵母细胞冷冻技术是生殖医学的一大热点,未成熟卵母细胞冻存是保存女性生育力的重要方法,具有广阔的临床应用前景。卵丘细胞与卵母细胞共处同一微环境,二者通过缝隙连接进行复杂的双相调控和物质交换,目前已知卵丘细胞在人类未成熟卵母细胞体外成熟过程中具有重要作用,但在冷冻过程中的作用尚无定论。随着玻璃化冷冻技术的发展,卵丘细胞对未成熟卵母细胞冷冻的影响有了较多的研究,卵丘细胞可减缓冷冻剂渗透速率,从而避免细胞渗透压的突变对未成熟卵母细胞的损伤,提高细胞冻融后存活率和受精率并支持未成熟卵母细胞的体外成熟。就卵丘细胞在人类未成熟卵母细胞玻璃化冷冻保存中的作用进行综述,为癌症化疗、延迟生育年龄等需进行的未成熟卵母细胞冷冻提供重要的理论基础及可靠的实验依据。     

HIV-1VN Jurkat细胞株HIV-1病毒分泌动力学及培养优化的研究

目的：研究HIV-1VN Jurkat细胞株HIV-1病毒分泌动力学及新生小牛血清对细胞生长及病毒产量质量的影响。方法：通过对HIV-1VN Jurkat细胞分泌病毒的滴度、细胞活力指数、活细胞密度、病毒比分泌速率和细胞比生长速率进行观察来评价其动力学；通过对抗原纯度的变化来说明不同浓度的新生小牛血清对分泌的病毒质量的影响。结果：HIV-1VN Jurkat细胞分泌病毒的滴度与细胞活力指数、活细胞密度呈现正相关性；病毒滴度、病毒比分泌速率与细胞比生长速率不相关，随着小牛血清浓度的增加，抗原纯度有下降趋势。结论：细胞生长对数期几乎同步出现病毒分泌对数期，小牛血清浓度为9％～12％的培养基是最佳的培养条件。     

卵母细胞玻璃化冷冻损伤相关因素及进展

卵母细胞玻璃化冷冻损伤的机制及影响因素的研究是近年来关于生殖能力贮存的研究热点。近年来随着细胞生物学及分子生物学的迅速发展,对卵母细胞冷冻损伤的影响因素研究取得了巨大进展。综述冷冻保护剂、卵母细胞特点及发育阶段、降温速率、卵母细胞的外围结构、冷冻方法等因素对卵母细胞冷冻损伤的影响机制及降低卵母细胞冷冻损伤的最近进展。     

非程序性冷冻仪冻存人胚胎干细胞的方法

在人胚胎干细胞（human embryonic stem，hES）的研究中，有效的冻存方法非常重要。目前，冷冻Es细胞主要有两种方法：慢速冷冻一快速复温和超快速玻璃化法。慢冻法需要昂贵的程序性冷冻仪，而玻璃化法步骤繁琐且只能冻存小量细胞．不能满足临床应用。因此，本研究旨在寻找非程序性冷冻仪冻存大量hES细胞的方法。     

胚胎玻璃化冷冻研究进展

玻璃化冷冻胚胎具有简便、迅速、经济、避免冰晶形成等优点,受到越来越广泛的关注。大量实验研究及初步临床应用结果表明,应用玻璃化冷冻技术能够保存多个种属不同发育阶段的胚胎。但是,目前玻璃化冷冻胚胎尚不成熟,应用合理的玻璃化冷冻溶液以及提高玻璃化冷冻速率等均是影响玻璃化冷冻胚胎的重要因素。     

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines

IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated.     

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency.Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log₁₀(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 10⁶ cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log₁₀(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.     

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost.Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10⁶ cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process.Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic.The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10⁶ cells/mL.     

消毒剂对脊髓灰质炎病毒灭活效果检测方法的初步探讨

[目的 ]　通过对试验细胞的选择 ,对作为干扰物的牛血清浓度的选择 ,用戊二醛和脊髓灰质炎Ⅰ型病毒 (PV-I)作为试验材料 ,探讨消毒剂灭活病毒效果检测方法的可行性。　 [方法 ]　采用悬液法测定消毒剂对病毒灭活效果。　 [结果 ]　发现选用Vero细胞比RD细胞更耐受消毒剂对细胞的毒性 ;不同浓度的牛血清作为有机物干扰戊二醛对PV -I的消毒效果有一定的影响。　 [结论 ]　细胞的维持时间和干扰物浓度的选择与试验的可靠性密切相关     

建立肾综合征出血热双价纯化疫苗人用疫苗生产传代细胞系Vero细胞库的研究

目的　建立HFRS双价纯化疫苗生产用三级细胞库 ,确保Vero细胞作为病毒培养基质的安全性。方法　以 2 0 0 2年版《中国生物制品规程》对生物制品生产用传代细胞系的要求 ,对Vero细胞进行核型分析、致肿瘤性等一系列基础性试验。结果　该生物所保存的Vero细胞未污染细菌、支原体 ,致肿瘤试验符合2 0 0 2年版《中国生物制品规程》要求 ,染色体核型分析结果为保持有原细胞学核学特征。结论　该所保存的Vero细胞 1 2 8代可以作为HFRS双化纯化疫苗生产的原始细胞库细胞     

Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells

Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 virus in Vero cells. DEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. Two of these eight recombinant viruses expressing distinct mutations in NS3 were both restricted in replication in the brains of suckling mice. In contrast, six recombinant viruses, each encoding individual mutations in NS4B (five) or in NS5 (one), were not attenuated in mouse brain. Recombinant viruses encoding various combinations of these Vero cell adaptation mutations did not demonstrate enhanced replication in Vero cells over that exhibited by the single mutations. Finally, addition of a subset of the above non-attenuating, adaptation mutations to a DEN2/4 chimeric vaccine candidate was found to increase the virus yield in Vero cells by up to 500-fold. The importance of these Vero cell adaptation mutations in flavivirus vaccine design and development is discussed.     

Comparative sensitivity of 4 different cell cultures in the isolation and identification of mumps virus]

Three different continuous cell lines (Am 57, Hela, Vero) and primary cell culture of human embryo kidney are compared with regard to their susceptibility for isolation of mumps virus from saliva and cerebrospinal fluid. For this purpose the Vero cell line appeared to be more suitable among the studied cell systems regarding the following reasons: it allows the highest percentage of mumps virus isolation. The cytopathic effect caused by mumps virus occurs comparatively rapidly in this cell culture. This cytopathic effect is typical enough to allow for a preliminary diagnosis of mumps virus.     

Vero-E6、MDCK、293细胞对SARS冠状病毒敏感性的研究

目的 研究Vero—E6、MDCK、293细胞对SARS冠状病毒的敏感性，为该病的病原学研究、诊断和疫苗制备奠定基础。方法 用Vero—E6、MDCK及293细胞对广东省部分SARS病人的咽拭子标本进行分离培养，并对分离物使用电镜、间接免疫荧光(IFA)及荧光定量聚合酶链反应(FQ—PCR)的方法分别检测细胞和(或)培养上清波中的病毒含量。结果 感染SARS病人的咽拭子标本后，Vero—E6细胞出现病变(CPE)最早，电镜中可观察到Vero—E6细胞中大量的病毒颗粒，IFA试验感染病毒的Vero—E6细胞与SAPS病人的恢复期血清呈强阳性反应，FQ—PCR结果显示Vero—E6感染细胞内的病毒含量10^8拷贝／ml，高于293细胞(10^6拷贝／ml)及：MDCK(104拷贝／ml)。结论 三种细胞对SARS相关病毒的敏感性不同，Vero—E6是SARS相关病毒最敏感的宿主细胞。     

汉坦病毒和恙虫病东方体复合感染的组织细胞培养研究

目的了解汉坦病毒（HV）和恙虫病东方体（Ot）在宿主体内共生情况.方法采用Vero E6细胞体外培养法研究HV、Ot在宿主细胞内共生特征.结果5组感染Vero E6细胞体外培养检测结果发现：先后、同时和单纯实验感染的5组细胞中,HV和Ot先后、同时接种感染组在细胞传至第3代时检测均见有HV和Ot的双重感染,阳性率随传代次数增加而增加.而HV和Ot单纯分别感染组细胞传第2代时即可检测到HV和Ot,阳性率亦随传代次数的增加而增加.在传代培养中单纯感染组的阳性率均显著高于先后和同时感染组.其结果均用PCR和RT-PCR进行了基因鉴定.结论研究结果提示HV和Ot可在同一宿主细胞内共生,在感染初期有相互抑制作用;对HV和Ot的流行病学有一定的理论意义.     

Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells

To confirm the safety of oral poliomyelitis vaccine (OPV) cultured in Vero cells, the genetic stability of cultured polio vaccine viruses was analysed by MAPREC (mutant analysis by PCR and restriction enzyme cleavage). The rates of mutant accumulation of the viruses passaged in Vero cells under a low multiplicity of infection (MOI) condition (approximately 10(-3.5)CCID50/cell; the same as under usual OPV production conditions) were higher than those passaged in secondary cultured monkey kidney cells. However, the rates of mutant accumulation were restrained when the viruses were cultured under a high MOI condition (approximately 10(-1.5)CCID50/cell) in Vero cells. Furthermore, neurovirulence of the passaged viruses in pollovirus susceptible transgenic mice PVR-Tg21 was shown to correlate highly with the results of MAPREC. It is expected that our results will contribute to the large scale preparation of safe and effective OPV using Vero cells.