细胞悬浮培养技术生产疫苗的研究进展

传统的疫苗生产方法受很多因素的限制,为了适应扩大化工业生产的需要,细胞悬浮培养技术已经在工业生产上日益成熟,本文就此项技术中关于悬浮培养的细胞性能、细胞悬浮培养驯化的方法、悬浮培养细胞培养基的优化、生物反应器种类等要素进行概述,并对此技术在疫苗生产中的应用作了展望。     

动物细胞悬浮培养技术研究进展

细胞悬浮培养是利用生物反应器大规模培养动物细胞生产生物制品的核心技术,是当前国际上生物制品生产的主流模式。作者就微载体的发展、各种生物反应器的基本原理及应用状况、悬浮培养技术存在问题、中国悬浮培养技术产业化存在的挑战和展望等作一综述。     

圆环疫苗悬浮培养?有专家指其炒作

正近年来,细胞悬浮培养技术逐渐被世人所知。不少生产厂家以此作为疫苗卖点,宣称采用悬浮培养新工艺,较之转瓶培养抗原含量更高、效果更好。不过不少专家指出,悬浮培养技术难点在于细胞驯化,并非所有细胞都适合悬浮培     

悬浮培养工艺与转瓶培养工艺的比较分析

采用反应器全悬浮培养BHK21细胞生产口蹄疫病毒与微载体悬浮培养Vero细胞生产狂犬病毒分别与相应的转瓶培养工艺生产案例对比分析,比较悬浮培养工艺与转瓶培养工艺的生产效益。分析显示,与转瓶培养工艺相比,反应器悬浮培养工艺获得的细胞密度、病毒效价、产品的产量和质量明显提高,生产时的能耗和劳动力需求明显降低。结果表明悬浮培养工艺的生产效益明显高于转瓶培养工艺,适宜于国内生物制品工业化生产的升级换代。     

BHK-21细胞悬浮驯化及对新城疫病毒悬浮培养工艺研究

为建立新城疫病毒在BHK-21细胞的无血清全悬浮培养工艺以获得高滴度和高纯度的新城疫悬浮培养抗原,通过悬浮培养驯化和筛选获得了形态良好、稳定传代的BHK-21-sc悬浮细胞株;该细胞以初始密度0.5×10~6 cells/mL接种,培养72 h可增殖到6×10~6cells/mL,细胞活率达95%。以5 L生物反应器悬浮培养BHK-21-sc细胞,对鸡新城疫病毒La Sota株的接毒剂量、TPCK胰酶添加浓度、病毒培养温度、收获时间等工艺参数进行了摸索和优化;并在5L-16L-50L生物反应器中进行逐级放大,以优化后的鸡新城疫悬浮培养工艺进行3个批次病毒悬浮培养。最终确定鸡新城疫病毒La Sota株接种BHK-21-sc悬浮细胞株的悬浮培养工艺:BHK-21-sc细胞悬浮培养的第3天按照感染复数(multiplicity of infection,MOI)为0.005接种病毒,并添加终浓度为5μg/mL的TPCK胰酶,于33℃培养72 h后收获病毒液。应用该悬浮培养工艺在5、16、50 L反应器上悬浮培养BHK-21-sc悬浮细胞株生产鸡新城疫病毒HA滴度不低于9log2,病毒含量不低于10~(6.0)TCID_(50)/0.1mL。表明BHK-21-sc细胞无血清全悬浮生产鸡新城疫病毒工艺稳定,可以实现逐级放大和规模化生产。     

ST贴壁细胞悬浮驯化及应用

为将ST贴壁细胞通过自主驯化,使其能在ST-S细胞无血清培养基中悬浮生长且能稳定传代,在摇瓶中实现高密度生长,并应用于猪伪狂犬病毒(PRV)悬浮培养,经ST-A低血清培养基适应培养,对一株贴壁的ST细胞进行了无血清的全悬浮驯化,并将PRV在悬浮细胞中连续盲传培养。结果显示:ST悬浮细胞能在无血清培养基中传代,培养48 h至第3代后,所能达到的最终细胞密度为3.00×10^(6 )cells/mL以上;PRV连续培养至第5代,毒价可达到109.0 TCID50/mL。结果表明,用专用培养基可使ST贴壁细胞实现悬浮驯化,并可应用于PRV悬浮培养。本研究为获得高密度ST悬浮细胞和提高PRV增殖效率奠定了技术基础。     

动物细胞悬浮培养技术在兽用疫苗生产领域中的应用

近年来,动物细胞悬浮培养技术备受关注,该技术已广泛应用于各类生物制品及兽用疫苗的研究和生产过程中。细胞悬浮培养生产兽用疫苗既能降低成本, 也能提高产品质量。以生物反应器技术为基础的细胞悬浮培养技术平台正逐步被建立起来且日趋成熟,成为推动兽用疫苗生产快速发展的主要动力。文章介绍了细胞悬浮培养技术,并就该技术在兽用疫苗生产中的应用进行了论述。     

首创传代细胞悬浮培养技术高效价猪瘟疫苗问世

<正>不久前,惠中集团洛阳普莱柯生物工程有限公司经过3年多攻关研究,利用国际最先进的传代细胞悬浮培养技术,率先在国内实现了猪瘟传代细胞悬浮培养重大工艺创新,填补了国内基于传代细胞悬浮培养技术生产高效价猪瘟疫苗之空白,解决了我国养猪业之急需。     

国内首创传代细胞悬浮培养技术高效价猪瘟疫苗问世

惠中集团洛阳普莱柯生物工程有限公司经过3年多攻关研究，利用国际最先进的传代细胞悬浮培养技术，率先在国内实现了猪瘟传代细胞悬浮培养重大工艺创新，填补了国内基于传代细胞悬浮培养技术生产高效价猪瘟疫苗之空白，解决了我国养猪业之急需。     

国内首创传代细胞悬浮培养技术高效价猪瘟疫苗问世

中国畜牧兽医报：不久前,惠中集团洛阳普莱柯生物工程有限公司经过3年多攻关研究,利用国际最先进的传代细胞悬浮培养技术,率先在国内实现了猪瘟传代细胞悬浮培养重大工艺创新,填补了国内基于传代细胞悬浮培养技术生产高效价猪瘟疫苗之空白,解决了我国养猪业之急需。     

Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.     

Immunocytochemical and morphometric studies on the pars distalis of the golden hamster from perinatal to senile stage

Six cell types of the pars distalis were studied from perinatal to senile stage in the golden hamster in combination with morphometric analysis and immunocytochemistry. GH cells occupied only a small area at 15 days of gestation and increased remarkably after birth to occupy a significantly larger area in the young adult than that in the other stages. PRL cells first appeared at 3 days after birth and increased rapidly thereafter to be distributed throughout the pars distalis. They always occupied a larger area in females than in males from 3 weeks after birth onward. ACTH cells were distributed mainly in the peripheral region of the pars distalis. They were dominant at 15 days of gestation, but relatively decreased after birth. The gonadotrophic cells were divided into LH cells and FSH cells. About 70% of LH cells were also immunoreactive to anti-FSH serum. LH cells occupied a larger area than that of FSH cells in every stage examined. TSH cells occupied the second largest area at 15 days of gestation, but rapidly decreased at 3 weeks after birth to occupy the smallest area among the immunoreactive cell types throughout the life-span. The chronological changes in the percentages of all cell types constituting the pars distalis seemed to reflect the activity of these cell types at a given stage.     

Regional Specialisation of the Ganglion Cell Density in the Retina of the Native Duck (Anas platyrhynchos) of Bangladesh

In this study, retinal whole‐mount specimens were prepared and stained with 0.1% cresyl violet for the ganglion cell study in the native duck (Anas platyrhynchos). The total number, distribution and size of these cells were determined in different retinal regions. The mean total number of ganglion cells was 1 598 501. The retinal area centralis had the highest ganglion cell density with 11 200 cells/mm². Number of ganglion cell bodies was the highest in temporal area, followed by dorsal, nasal and ventral areas. Ganglion cell size ranged from 5.25 to 80 μm². In the temporal and nasal region, most of the cells were ranged from 15 to 25 μm², and in the dorsal and ventral region, most of the cells were ranged from 12 to 25 μm². There was a marked trend for the retinal ganglion cell size to increase as the population density decrease towards the periphery. A population of small ganglion cells persisted into the central area just above the optic disc and the largest soma area was in the ventral zone of the retina. Thus, the specialisation of ganglion cell densities and their sizes support the notion that the conduction of visual information towards the brain from all regions of the retina is not uniform, and the central area is the fine quality area for vision in native duck.     

Electron microscopy of mast cells in the venous wall of canine liver

Although many mast cells locate under the endothelial layer along the sublobular veins in canine liver, the cell function remains to be fully defined. To establish the nature of the canine mast cell, the mast cells were examined by electron microscopy. A few monocytes contacted with luminal surface of endothelial cells under which mast cells situated. To confirm the chemotaxis of monocyte by hepatic mast cells, the hepatic venous vessels were treated with a histamine releaser (compound 48/80). The monocytes invaded into the subendothelial layer and extended their pseudopodium to the degranulated mast cells. It presumes that some mediators within the mast cell granules might act as a chemotactic substance to the monocyte. On the contrary, mast cells were migrating from subendothelial layer to venous lumen under normal condition. The migrating mast cell showed strong acid phosphatase reaction in their granules. It suggests that the granules of migrating mast cell became visible to acid phosphatase activity by a physical force such as contact stimulation, and that a part of mast cells remigrate from the venous wall to other places by the blood flow. Furthermore, hepatic mast cells were revealed to contain both endothelin-1 and histamine in their granules by immunocytochemistry. As these substances have an activity of stronger venous constriction, it seems that the mast cells play an important role in the blood flow regulation of the canine liver, mast cell, monocyte.     

The effect of the lactation period on the cell content of sheep milk]

Milk samples of 201 ewes were examined in 6 week intervals during a complete lactation period. Those samples were analyzed for the presence of pathogenic bacteria and the somatic cell count was determined. Besides, the California Mastitis Test (CMT) was performed and the udder was clinically examined. The cell counts were found to depend on the lactation period. During 6 weeks following parturition the cell count was 63,000 cells/ml. This number decreased towards the 24th week of lactation to 32,000 cells/ml. At the end of lactation this value increased again to 425,000 cells/ml. The median value of ewes with normal udder health was 56,000 cells/ml milk. For samples from which pathogenic bacteria were isolated this value was 159,000 cells/ml. The most frequent pathogens isolated from the milk samples were coagulase-negative cocci (59.6% of bacteriologically positive samples), the median number being 88,000 somatic cells/ml in these sheep. Coagulase-positive cocci were isolated in 25.3% of the samples, the median value of the cell count was 295,000 cells/ml. In 12.1% of the samples streptococci were found. The median value was 167,000 cells/ml. From the remaining 3.0% of bacteriologically positive samples Pasteurellae, E. coli and Actinomycetae were isolated. The median value of the somatic cell count was 184,000 cells/ml. We consider coagulase-positive cocci therefore as the most pathogenic bacteria for the ovine udder.     

Epithelium lining the rostral portion of the porcine nasal mucosa

Epithelial tissues from the rostral half of the pig nasal cavity were prepared for light and electron microscopy. The predominant epithelial cell type at the luminal surface was cuboidal with surface microplicae or microvilli and a multilobate nucleus. Pinocytotic vesicles were a common feature of the adluminal cytoplasmic zone of these superficial cuboidal cells. Other cell types included basal cells, intermediate cells and, occasionally, goblet and ciliated cells. Basal cells contained vesicles located adjacent to the basal lamina. This transitional mucosa may be specialised for sampling substances from the luminal surface preliminary to releasing cytokines and, or, presenting immunogens to intraepithelial lymphocytes.     

Seasonal variation of bulk milk somatic cell counts in UK dairy herds: investigations of the summer rise

Individual cow somatic cell count (SCC) patterns were explored over a one year period in 33 dairy herds to investigate the reason for a summer rise in bulk milk somatic cell counts (BMSCC). Cow test day somatic cell counts were categorised according to the magnitude of change since the previous test day reading, to examine which categories were responsible for the summer increase. Multilevel models using Markov chain Monte Carlo methods were specified to estimate the number of somatic cells/ml produced by different cell count categories. Stage of lactation and parity were accounted for in the models. There was an increase in the proportion of cows that remained above 200,000 cells/ml for two consecutive recordings in summer and this group of cows were responsible for 70.8% of the increase in somatic cells/ml produced from May to September compared with October to March. There was no evidence that a greater new infection rate (somatic cell counts moving from below 100,000 cells/ml to over 200,000 cells/ml) contributed to the increased summer bulk milk somatic cell counts. There was no indication that a general small increase in all somatic cell counts played an important role in the increased summer somatic cell counts. Markov chain Monte Carlo methods provided a valuable and flexible platform for parameter estimation in reasonably complex multilevel models.     

Induction of T cell anergy by the treatment with IL-10-treated dendritic cells

In order to apply for reducing graft versus host disease in allogeneic stem cell transplantation, the study concerning the induction of specific T cell anergy was designed. Normal allogeneic lymphocytes, which were co-cultured with IL-10-treated immature dendritic cells in the first mixed leukocyte culture (MLC), were cultured with mature dendritic cells of the same origin as IL-10-treated immature dendritic cells in the second MLC. By co-culturing with IL-10-treated immature dendritic cells, the response of normal lymphocytes to mature dendritic cells cultured from the same individual as that of IL-10-treated dendritic cells was markedly reduced, compared with the lymphocytes cultured with non-treated dendritic cells or IL-10-treated dendritic cells from a third party individual. The present study demonstrated that antigen specific T cell anergy was generated by priming allogeneic lymphocytes with IL-10-treated immature dendritic cells. These data suggested the applicability of IL-10-treated recipient dendritic cells for the induction of recipient cell-specific donor T cell anergy in donor graft.     

表达猪瘟兔化疫苗株E0蛋白的PK-15细胞系构建

将克隆到pGEM T-easy载体中的E0基因双酶切回收后，连接到增强型绿色荧光蛋白（EGFP）中，利用Lipofectamine^TM2000将重组子转染PK-15、BHK-21、VERO 3种细胞，直接在荧光显微镜下用蓝光激发进行观察，在3种细胞中都可以观察到绿色荧光，而且在转染后的24、48、72 h 3个时间点上，观察到的荧光细胞数量逐渐增多，在72 h时荧光细胞的数量在3种细胞中都达到最多，总的来看，在PK 15中荧光细胞的数量最多。通过对细胞荧光的定位发现，重组质粒的荧光主要分布在胞浆内，而且细胞核周围的荧光较强，胞核与胞浆的界限明显，尤其是在PK-15细胞和VERO细胞中这种现象尤为明显。未携带E0目的DNA的pEGFP-N1 Vector转染3种细胞后，都可以观察到绿色荧光，但是整个细胞中是均匀出现荧光的。经酶切、PCR、单抗间接免疫荧光检测，PK-E0细胞中有大量的HCLV的E0表达，单纯的PK-15细胞中没有E0的表达。HCLV株在PK-E0细胞中从48h开始到72h的滴度比在PK-15细胞中的滴度要高。证明PK-E0细胞中E0蛋白的大量表达增加了HCLV在细胞中的复制。