Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 10⁶ cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 10⁵ cells/mL resulted in a virus titer higher than 10⁷ FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 10⁷ FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca⁺⁺ and Mg⁺⁺. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.     

Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine

Peste des Petits ruminants (PPR) is considered as one of the major constraints to the productivity of small ruminants in Africa and Asian countries. Currently PPR control is done by vaccination with an attenuated PPR strain (Nigeria 75/1) produced in monolayers of Vero cells grown in roller bottles or static flasks. This work focuses on the production of a PPR vaccine strain using stirred conditions as an advanced option for process scale-up. Non-porous microcarriers (Cytodex-1) were used to support Vero cell growth in suspension cultures. The use of Ex-Cell medium could improve cell specific productivities obtained with standard serum containing medium, independently of the type of system used, i.e. static as well as suspension stirred cultures. As an alternative, several cell lines adapted to grow as single cells in suspension (CHO-K1, BHK-21A and 293) and another anchorage-dependent (MRC-5) were evaluated in their capacity to produce a PPR vaccine. BHK-21A and 293 cells grown as single-cell suspension in serum free medium were both suited to produce PPR vaccine with productivities similar to Vero cells, namely 10(6)TCID(50)/mL. However, for the 293 cells, these results were only obtained 2-3 days later. CHO-K1 and MRC-5 cells have shown not to be suitable to adequately produce this virus. These results provide further insights into the feasibility of applying microcarrier cell culture technology to produce PPR vaccine in Vero cells as well as in the alternative use of single-cell suspension cultures of BHK-21A, significantly simplifying the existing production process.     

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM+10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85x10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2x10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6x10(6) cells/mL was obtained when the cells were grown on 3g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4x10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5x10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38x10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium.     

Purification of rabies virus produced in Vero cells grown in serum free medium

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.Rabies virus purification using the monolithic column resulted in the highest antigen recovery yield, equal to 94%. Capto Core 700 showed a lower yield, about 84%; whereas the purification yield by zonal centrifugation was equal to 60%. In terms of host cell residual DNA removal, zonal centrifugation was the most efficient method; the removal yield was equal to 88.5%; elimination of host cell DNA was slightly lower when using the monolithic CIM-QA (equal to 73%). Whereas Capto Core 700 showed the lowest level (49.2%). Host cell protein removal varied between 92.6% for the monolithic column and 78.6% for the zonal centrifugation. Capto Core 700 eliminated 86.5% of HCP.     

风疹病毒山东地方株的分离与鉴定

目的 了解山东省风疹病毒流行情况，进行风疹野病毒山东地方株的分离、鉴定。方法 2001～2002年，从我省的济宁、淄博、济南、德州市风疹爆发流行中，采集53份具有风疹典型症状患者的咽拭子，经Vero，RK-13，BHK-21，MK2细胞分离培养，选出有明显的细胞病变株。结果 在Vero细胞上对HSV-1病毒攻击呈干扰现象；风疹免疫荧光阳性者；共分离出9株风疹病毒山东地方野毒株(SDRV)，代码为SDRV1、2、3、4、5、6、7、8、9。结论 细胞用于风疹病毒的分离培养，免疫荧光实验可作为风疹病毒鉴定、诊断的重要方法。     

Use of MDCK cells for production of live attenuated influenza vaccine

To develop a cell-based live attenuated influenza vaccine (LAIV) manufacturing process, several different cell lines were evaluated by comparing the titer of viruses after infection with LAIV strains. While several cell lines have been reported to support influenza virus replication, the degree of replication and the ability to support replication of LAIV strains have not been systematically examined. MDCK cells, which have been considered as potential substrates for influenza vaccine production were evaluated in addition to Vero, MRC-5, WI-38 and FRhL cells. MRC-5, WI-38 and FRhL cells produced low to moderate titers of virus with titers equal or below 5.0 log₁₀ TCID₅₀/mL. Both Vero and MDCK cells could support a higher level of virus replication for certain strains, however, Vero cells only produced high titers when grown in the presence of serum. MDCK cells supported high levels of vaccine virus production for multiple different LAIV subtypes in both serum containing and serum-free media. These results suggest that MDCK cell-based production can be used as an alternative production platform to the currently used egg-based LAIV production system.     

Rabies immunization of red foxes (Vulpes fulva) with vaccine in sausage baits.

Foxes were orally vaccinated against rabies with two modified live vaccine viruses, ERA/BHK-21 and PRI strain, in sausage baits. The baits proved acceptable to caged foxes and effectively delivered immunizing doses of vaccine. Antibody profiles on the vaccinated foxes and intramuscular challenge with "street" rabies virus showed the ERA/BHK-21 vaccine to be more effective than the PRI vaccine. The addition of a stabilizer helped maintain virus titers when the baits were subjected to high (35 C) temperature.     

HEP-Flury株病毒辅助质粒包装CTN株病毒基因组拯救狂犬病病毒

目的 以CTN株狂犬病病毒全长基因组cDNA重组质粒(pCTN-GFP)和HEP-Flury株病毒N、P、L辅助质粒共转染拯救具有活性的CTN株重组狂犬病病毒(CTN-GFP).方法 将CTN株狂犬病病毒全长基因组分4段进行扩增,体外连接扩增片段并克隆入表达载体中,构建CTN株狂犬病病毒全长基因组cDNA重组质粒,基因组ψ基因被标识基因GFP取代,通过与HEP-Flury株病毒N、P、L3个辅助质粒共转染BHK-21细胞,拯救能够稳定表达GFP的重组病毒CTN-GFP.结果 成功扩增全长基因组的4个基因片段,并将其克隆入表达载体,构建了CTN株狂犬病病毒全长基因组cDNA重组质粒pCTN-GFP,经酶切鉴定和序列测定证明pCTN-GFP为正确克隆,可直接用于病毒拯救.将pCTN-GFP与HEP-Flury株病毒辅助质粒共转染BHK-21细胞4d后,成功拯救出CTN株重组狂犬病病毒,重组病毒能够表达标识基因GFP,荧光显微镜下可直接观察到标识基因GFP的表达,设计跨GFP和L基因的特性引物对重组病毒进行RT-PCR检测,结果证明此病毒是从克隆的质粒中拯救的重组病毒.结论 HEP-Flury株病毒的结构蛋白能够包装并转录CTN株病毒的基因组RNA.     

In vitro and in vivo effects of West Nile virus propagated in cells of different passage

Tissue cultures are used to propagate viruses for use in mosquito infection studies and to detect live virus in field-collected specimens. Microscopic evaluation of cytopathic effects is used to visualize virus presence. In this study, both low-passage (LP; n = 35) and high-passage (HP; n = 218) lines of African green monkey kidney (Vero) cells were infected with West Nile virus, and virus growth kinetics were quantitated over different incubation periods (IPs) (2, 6, 10, 24, 48, 72, and 96 h postinfection). Virus titers were significantly (P < 0.05) higher in the HP compared with LP line 24, 48, and 96 h postinfection. No differences were observed in plaque morphology between the LP and HP lines. Culex pipiens quinquefasciatus were fed infectious blood meals created using supernatant from the 2 lines and maintained at 28 degrees C for a 12-day IP. Although the virus dose was higher (P < 0.05) in the HP compared with the LP line, there were no significant differences in mosquito infection or dissemination rates at the end of the IP. The significance of these observations for assessing virus presence and pathogenicity is discussed.     

狂犬病aG毒株适应3aG-V生产株的特性研究

目的研究狂犬病固定毒Vero细胞适应株3aG-V生产株的生物学特性。方法观察毒株形态、培养条件、致病性、免疫原性、毒力试验及其在中枢神经系统是否形成尼氏小体。结果狂犬病aG固定毒3aG-V株具有抗原性好,培养产毒量高,保持有aG固定株弱毒性、传代稳定、无变异的特性。结论狂犬病aG固定毒3aG-V株可作为替代地鼠肾细胞狂犬病疫苗aG毒株,用于Vero细胞培养病毒生产出毒液毒力高、灭活后效力高、安全性好的纯化Vero细胞狂犬病疫苗的生产用疫苗株。     

Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells

The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging.Here we present a production platform based on BHK-21 suspension (BHK-21_SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1–48 PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV^PE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21_SUS cells with ZIKV^PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9 × 10³ PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2 × 10⁷ cells/mL, and virus titers of 3.9 × 10⁷ PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3 PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.     

RC-IAL cell line: sensitivity of rubella virus grow

OBJECTIVE: The rapid growth of the rubella virus in RC-IAL2 with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.     

过氧化负荷对正常及恶性成纤维细胞的抗氧化物和脂质过氧化物水平的影响

采用细胞培养及自由基生化方法,研究正常和过氧化条件下,幼年仓鼠肾成纤维细胞(BHK-21/C13)及其多瘤病毒转化株(BHK-21/PyY)的抗氧化物和脂质过氧化物水平,发现:非转化株的抗氧化物水平,除谷胱甘肽S-转移酶(GST)和谷胱甘肽还原酶(GSHR)外,谷胱甘肽过氧化物酶(GSHPx)、还原型谷胱甘肽(GSH)、硒水平均高于转化株(BHK-21/PyY);培养基中加入维生素E,非转化株比转化株摄入更多的维生素E;而转化株的内源性丙二醛(MDA)的产生多于非转化株。在铁催化的过氧化条件下,除硒、GST和GSHR外,二株细胞内上述抗氧化物指标都显著下降,MDA产生显著增加。除GSHPx外,非转化株的这种上升和下降的变化幅度均大于转化株。提示:自由基代谢水平在正常及恶性细胞之间,至少在上述二株细胞之间有所不同;正常细胞对过氧化的敏感性高于恶性细胞。     

精制Vero细胞狂犬病疫苗的研制及免疫学效果观察

目的　以Vero细胞为基质，研制新一代精制Vero细胞狂犬病疫苗。方法　以CTN-1V10为生产毒株，以＜150代Vero细胞为培养基质，采用转瓶旋转培养，按不同时间收获病毒液，经过澄清、浓缩、纯化、灭活制成精制Vero细胞狂犬病疫苗。从以此工艺制备的疫苗中选取一批做为免疫学效果观察的受试疫苗。按暴露后免疫程序接种63人，其中受试疫苗接种33人，法国维尔博狂犬病疫苗接种30人，观察不良反应和测定中和抗体。结果　新研制的精制Vero细胞狂犬病疫苗各项指标完全符合WHO的有关质量要求。两种疫苗全程免疫后中和抗体阳转率均为100%，中和抗体受试组为11.94IU/ml;维尔博疫苗对照组为11.69IU/ml。结论　精制Vero细胞狂犬病疫苗不但制造工艺合理，而且副反应轻微，免疫原性良好。     

乙脑病毒SA14-14-2减毒株病毒外源因子污染检测分析

目的 检测乙脑病毒SA14- 14 -2减毒株的病毒外源因子.方法 用乙脑病毒免疫血清(阳性)将乙脑病毒SA 14 - 14 -2减毒株中和后,采用中和后的样品分别感染指示细胞(3T3、NRK、C3/36细胞),直接培养观察及红细胞吸附试验;中和后样品感染小鼠细胞系(3T3)培养14d的上清液再接种指示细胞(MRC -5、Vero、BHK21、3T3)培养观察及红细胞吸附试验;同时将流感病毒接种MDCK细胞,作为试验阳性对照.结果 乙脑病毒SA 14 - 14-2减毒株中和后的样品接种指示细胞(3T3、NRK、C6/36细胞),直接培养观察7d、14 d,细胞形态正常,无细胞病变征兆,红细胞吸附试验为阴性;中和后样品感染小鼠细胞系(3T3)培养14 d的上清液再接种指示细胞(MRC -5、Veto、BHK - 21、3T3)培养观察7d、14 d,细胞形态正常,无细胞病变征兆,红细胞吸附试验为阴性.结论 乙脑病毒SA14-14-2减毒株中未检测到人源、猴源、鼠源及其他病毒的污染,符合《WHO Technical Report Series,No.910,2002》的质量标准,可安全地用于乙脑减毒活疫苗的生产.     

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.     

Immunogenicity and efficacy of an in-house developed cell-culture derived veterinarian rabies vaccine

The efficiency of an inactivated tissue culture rabies vaccine produced on BHK-21 cells, according to an in-house developed process, was evaluated and compared to a commercial cell-tissue culture vaccine (Rabisin). Fifteen experimental dogs from local common breed were duly conditioned during a quarantine period, then vaccinated via the subcutaneous route with 1 ml of either the tissue culture vaccine developed in-house or the commercial vaccine Rabisin. The immune response of each dog was monitored for 162 days. Serum-neutralizing antibodies titers to rabies virus were determined by the rapid fluorescent focus inhibition test (RFFIT) which confirmed the strong response of dogs to both vaccines except one dog in the Rabisin group. The dogs were then challenged in the masseter muscle with a rabies street virus of canine origin. All vaccinated dogs except the single dog in the Rabisin group that failed to respond to the vaccine, survived the challenge. In contrast, 80% of animals in the control non-vaccinated group, developed rabies and died. A field vaccine trial was also conducted: 1,000 local dogs living in field conditions received one subcutaneous dose of the locally developed vaccine. Serum neutralizing antibody titers to rabies virus was determined by RFFIT at days 0, 60 and 360. Mean rabies neutralizing antibody titers were equal to 0.786, 3.73 and 1.55 IU/ml, respectively. The percentage of dogs with a neutralizing rabies antibody titer higher than the 0.5 IU/ml mandated WHO threshold, was 30%, 91.4% and 87.5% at day 0, 2 months and 1 year post-vaccination, respectively. These data demonstrate the efficiency of the in-house developed vaccine produced on BHK-21 cells in both experimental and field conditions and support its use in dog mass vaccination campaigns.     

Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development

Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities. The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs. Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production. In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells. High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells. For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection. A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production. The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice. These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine.     

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.     

Vero-cell rabies vaccine produced using serum-free medium

A new rabies vaccine was developed from Vero cells adhered to microcarriers, cultivated in a bioreactor in serum-free medium and infected with the PV/VERO-Paris rabies virus strain. The viral suspensions were concentrated by tangential filtration, purified by chromatography and inactivated with beta-propiolactone. In immunogenicity studies performed in mice immunized with three doses of the new vaccine (seven batches) and the commercial Verorab and HDCV, mean titers of neutralizing antibodies of 10.3-34.6, 6.54 and 9.36 IU/ml were found, respectively. The vaccine presented stability during 14 months at 2-8 degrees C, 30 days at 37 degrees C and 8 h at 45 degrees C. The use of serum-free medium facilitated the downstream process leading to residual cellular DNA values <22.8 pg per dose of vaccine in all produced batches. The effective immunogenicity induced in mice by this vaccine, the degree of purity of the product, the high antigen yield and the reduction of the cost of the product due to the virus production and purification processes, makes this technology very important for countries where rabies presents a great public health problem.