Vero细胞无血清培养基的研究进展

Vero细胞是世界卫生组织和《中国药典》认可的用于人用疫苗和动物疫苗生产的细胞系,Vero细胞无血清培养生产疫苗已成为当前的主要趋势。无血清培养的关键是设计符合Vero细胞贴壁特性和提高细胞密度的无血清培养基,这也是规模化培养的关键因素之一。Vero细胞无血清培养基的开发与使用一方面减少了对动物血清的依赖,提高了病毒性疫苗的质量安全;另一方面促进了无血清培养技术的发展与应用。现就Vero细胞无血清培养基的研究进展予以综述。     

无血清细胞培养在发热伴血小板减少综合征布尼亚病毒生长中的应用

目的建立无血清培养基培养Vero细胞制备发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的工艺。方法分别采用含10%牛血清的MEM(10%MEM培养基)和无血清M2培养基(SF-M2培养基)在方瓶中培养Vero细胞制备SFTSV,比较无血清与含血清培养基培养Vero细胞制备SFTSV在病毒滴度及病毒繁殖曲线之间的差异。在生物反应器里用无血清培养的方式进行工艺放大,收获病毒原液并进行检定。结果无血清培养的Vero细胞能够满足SFTSV培养需求,与含血清细胞培养相比,单位细胞病毒产量没有降低,达到30~60个活病毒/细胞。可以实现在生物反应器的工艺放大,病毒高峰时病毒滴度均在7.0lg PFU/m L以上。结论无血清细胞培养可以应用于SFTSV的培养,有利于降低疫苗生产过程中的纯化难度,提高疫苗安全性。     

Vero细胞作为基质的病毒性疫苗研制进展

使用传代细胞系Vero细胞生产脊髓灰质炎疫苗和狂犬病疫苗已有多年的历史。随着传代细胞系潜在致癌性的相关检测技术不断发展完善,消除了人们对其潜在致癌性的担忧,监管部门正逐步认可使用传代细胞系来生产病毒性疫苗。目前,Vero细胞被越来越多地用于生产病毒类疫苗,并用生物反应器工艺取代转瓶工艺,便于规模化培养。本文就Vero细胞作为基质研制的病毒性疫苗作一综述。     

重组HSV-Ⅱ病毒疫苗的微载体悬浮培养生产工艺研究

经过重组减毒的Ⅱ型溶瘤单纯疱疹病毒(rHSV-Ⅱ)在体外具有良好的溶瘤效果,并对小鼠体内B16R黑色素瘤内注射具有较高的疗效。重组HSV-Ⅱ病毒作为高效的新型溶瘤病毒疫苗有望用于肿瘤的临床治疗。Vero细胞是广泛应用于病毒疫苗生产的细胞基质,该细胞系对多种病毒敏感且产量高,其微载体培养安全,开发重组HSV-Ⅱ溶瘤病毒肿瘤疫苗的Vero细胞微载体反应器悬浮培养生产工艺具有重要的意义。以自主开发的低血清培养基为基础,对Vero细胞的反应器微载体球转球转移放大工艺及在位消化放大培养工艺进行了研究和比较,以新老微载体比3:1的比例成功实现了微载体球转球转移放大,建立了可实现至少四次/级连续放大的球转球转移放大培养工艺,可用于工业化生产过程。在此基础上,初步建立了以Vero细胞为基质的重组HSV-Ⅱ病毒疫苗反应器微载体无血清悬浮培养生产工艺,最大病毒滴度可达到6.62 lgTCID50/ml以上。为以Vero细胞为基质的无血清病毒疫苗规模化培养提供了一种简便、高效的工艺方法。     

Vero细胞和脊髓灰质炎病毒在无血清条件下的培养探究

目的：探索Vero细胞和脊髓灰质炎病毒在无血清条件下的最佳培养条件,为无血清培养Vero细胞生产脊髓灰质炎疫苗奠定基础。方法选择直接适应（直降组）和序贯适应（驯化组）两种无血清培养方法,观察Vero细胞和脊髓灰质炎病毒在无血清条件下的生长情况,并检测脊髓灰质炎病毒及其病毒滴度。结果 Vero细胞在两种无血清条件下均生长良好,其中驯化组细胞生长速度更接近对照组。以脊髓灰质炎病毒Sabin株Ⅰ型分别感染直降组、驯化组和对照组细胞的病毒滴度平均值分别为8．94、8．81和8．94 LgCCID50/mL;Ⅱ型病毒滴度平均值分别为8．84、8．25和7．94 LgCCID50/mL;Ⅲ型病毒滴度平均值分别为8．91、8．57和8．63 LgCCID50/mL;且3组的变异系数（ CV）均小于10％。结论 Vero细胞在无血清条件下生长良好,无血清培养的Vero 细胞可用作脊髓灰质炎疫苗生产的基质。     

组成型过量表达hTERT对Vero细胞体外培养生物学特性的影响

目的：考察组成型过量表达人端粒酶催化亚单位（hTERT）对Vero细胞在无血清培养体系中的细胞形态、生长和代谢的影响。方法：以组成型过量表达hTERT的Vero细胞系T1为研究对象,以活细胞密度和细胞活力为主要观察指标,结合细胞形态和贴附伸展动态,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养和悬浮培养体系中的细胞生长;以葡萄糖比消耗速率（qglc）、乳酸比生成速率（qlac）、乳酸转化率（Ylac/glc）和谷氨酰胺比消耗率（qgin）为反映细胞代谢的主要观察指标,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养的细胞代谢。结果：hTERT组成型过量表达在降低Vero细胞的贴附伸展能力和对血清的依赖程度的同时,提高了细胞无血清批次培养后期的细胞活力和活细胞密度,并赋予了T1非贴附依赖性生长的能力。hTERT组成型过量表达未对Vero细胞的代谢产生明显的影响。结论：hTERT组成型过量表达可降低Vero细胞的贴附生长依赖性和对血清的依赖程度,是有应用潜力的改良哺乳动物细胞体外培养性状的技术途径。     

组成型过量表达hTERT 对Vero 细胞体外培养生物学特性的影响

目的:考察组成型过量表达人端粒酶催化亚单位(hTERT)对Vero细胞在无血清培养体系中的细胞形态、生长和代谢的影响。方法:以组成型过量表达hTERT的Vero细胞系T1为研究对象,以活细胞密度和细胞活力为主要观察指标,结合细胞形态和贴附伸展动态,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养和悬浮培养体系中的细胞生长;以葡萄糖比消耗速率(qglc)、乳酸比生成速率(qlac)、乳酸转化率(Ylac/glc)和谷氨酰胺比消耗率(qgln)为反映细胞代谢的主要观察指标,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养的细胞代谢。结果:hTERT组成型过量表达在降低Vero细胞的贴附伸展能力和对血清的依赖程度的同时,提高了细胞无血清批次培养后期的细胞活力和活细胞密度,并赋予了T1非贴附依赖性生长的能力。hTERT组成型过量表达未对Vero细胞的代谢产生明显的影响。结论:hTERT组成型过量表达可降低Vero细胞的贴附生长依赖性和对血清的依赖程度,是有应用潜力的改良哺乳动物细胞体外培养性状的技术途径。     

Vero细胞微载体培养的化学成分明确无血清培养基的设计

目的：设计适用于Vero细胞微载体培养的化学成分明确无血清培养基。方法：以商品化的DMEM／F12合成培养基为基础培养基,应用Plackett—Burman实验设计和响应面分析法设计支持Vero细胞微载体培养的化学成分明确无血清培养基。结果：以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计考察10种培养基添加成分对Vero细胞生长的影响,确定了3种对Vero细胞生长起明显促进作用的培养基添加成分,为胰岛素、血清素和腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种支持Vero细胞贴附培养的无血清培养基（VERO—SFM—A）。在Bellco搅拌式培养瓶中采用VERO-SFM．A和Cytodex1微载体培养Vero细胞,细胞密度由接种时的4×10^5cells／ml增加到培养6d后的22．3×10^cells／ml,细胞活力保持在96％以上。结论：VERO—SFM—A能够有效地支持Vero细胞在微载体表面固定化生长并达到较高的细胞密度,具有实际应用于Vero细胞微载体规模化培养的应用潜力。     

肾综合征出血热双价纯化疫苗（Vero细胞）安全性的研究

在肾综合征出血热（HFRS）双价纯化疫苗（Vero细胞）的研制中,对病毒培养用Vero细胞致瘤性和试制疫苗的过敏原性进行了动物实验研究,结果表明,病毒培养用Vero细胞无致瘤性,三批试制疫苗无过敏原性。说明用Vero细胞为培养介质的HFRS双价纯化疫苗是安全的。     

流感病毒在Vero细胞系与MDCK细胞系增殖条件的比较

目的研究流感病毒H1N1及其他亚型在Vero细胞系和MDCK细胞系高效增殖的最适条件,比较两种细胞系对流感病毒的敏感性差异及影响敏感性差异的条件。方法在培养好的Vero细胞系与MDCK细胞系用不同的病毒感染复数(M.O.I)、胰酶浓度、病毒吸附时间、病毒维持液血清质量浓度等条件进行流感病毒在细胞上的增殖。结果在M.O.I为0.01接种流感病毒,吸附时间为1 h,胰酶质量浓度2μg/mL,血清质量浓度为8%时,流感病毒血凝素在MDCK细胞系可获得较高的滴度。结论 MDCK细胞系是适于流感病毒培养的细胞,它作为生产新型流感病毒疫苗的主要细胞基质需要进一步的研究。     

Stationary and microcarrier cell culture processes for propagating Japanese encephalitis virus

Inactivated mouse-brain-derived vaccines for Japanese encephalitis virus (JEV) have been used for many years. Recently, attempts have been made to employ cultured Vero cells to replace mouse brain tissues for developing cell-culture-derived vaccines that will be more suitable for worldwide usage. In this study, JEV replication processes in Vero and BHK cells and between stationary and microcarrier culture systems were investigated. Our results demonstrated that a stationary Vero cell culture system produced higher viral titers of JEV, including the Beijin-1 vaccine strain and the attenuated strain CH2195LA, than microcarrier culture did. BHK cells showed less significant differences in their replication kinetics between stationary and microcarrier cultures. Reducing serum concentration during infection led to an overall decrease of JEV production in Vero cells but an increase in BHK cells. By establishing a complete serum-free Vero cell culture, the microcarrier system resulted in a more than 4-log lowered yield compared to that of the stationary culture for JEV production. Thus, the stationary culture is the most efficient system for JEV production from cultured Vero cells.     

Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.     

Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion

Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.     

Screening of a high growth influenza B virus strain in Vero cells

Due to the insufficient supply of embryonated chicken eggs, the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus. The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines. However, most of the influenza viruses can not grow well in Vero cells. To develop a new influenza vaccine with Vero cells as a substrate, the virus needs to adapt to this cell substrate to maintain high growth characteristics. By serial passages in Vero cells, the B/Yunnan/2/2005va (B) strain was successfully adapted to Vero cells, with the hemagglutination titer (HAT) of the virus reaching 1:512. The high growth characteristic of this strain is stable up to 21 passages. The strain was identified by hemagglutination inhibition (HAI) test and sequencing respectively; the HA₁ gene sequence of the virus was cloned and analyzed. The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.     

Proteome analysis of virus–host cell interaction: rabies virus replication in Vero cells in two different media

The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to th...     

Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10(8) TCID(50)/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.