微载体浓度与细胞接种密度对ST细胞生长的影响

选择合适的微载体浓度、细胞接种密度以提高微载体利用率,优化微载体培养体系猪睾丸细胞(Swine testicle cells)的贴附生长与维持。使用DMEM补加10%血清、LSM(Low serum medium)两种培养基考察微载体浓度、细胞接种密度对细胞生长维持的影响,进而比较ST细胞在不同条件下对Cytodex1微载体的利用率。结果显示,使用LSM在T150方瓶中连续传代培养30d,平均比生长速率为0.626d~(0-1),是DMEM补加10%FBS培养基的1.15倍。选择10×10~5cells/mL细胞接种3g/LCytodex1搅拌瓶体系,最大细胞密度为38.3×10~5cells/mL,微载体利用率上升到58.8%。在灌注培养体系中培养ST细胞15d,最终细胞密度达到36.6×10~5cells/mL,扩增了13.6倍。微载体悬浮培养的使用一方面有利于ST细胞的贴附与生长,实现高密度生长,另一方面增加了微载体的使用成本,选择合适的微载体浓度、细胞接种密度,能够最大化利用微载体与培养基中的营养物质实现细胞的最优生长。     

MDCK细胞微载体培养条件优化研究

研究细胞接种量、搅拌转速和微载体浓度对MDCK细胞微载体培养时的影响,以合理优化MDCK细胞微载体培养最大增殖时期的最优条件,对疫苗和病毒分离具有重要意义,以期达到在疫苗和病毒分离领域提高生产效率。采用微载体培养MDCK细胞,在不同搅拌转速、微载体浓度和细胞接种量进行培养,每隔24 h取样计数,确定最优的培养条件。结果表明,细胞接种量在20个/球、搅拌转速45 r/min和载体浓度在2 g/L时,MDCK细胞的增殖较快,细胞密度较大,细胞的密度最大可达15.6×106个/mL,适合MDCK细胞增殖生长。     

H1N1流感病毒在微载体培养MDCK细胞上增殖的研究

目的探索MDCK细胞在微载体上的培养条件,并研究H1N1型流感病毒在MDCK细胞上的增殖条件。方法在微载体上培养好MDCK细胞上用H1N1型流感病毒在不同的病毒感染复数(MOI)、胰酶浓度两个关键的病毒增殖条件进行流感病毒在细胞上的增殖研究。结果微载体质量浓度为6 g/L时,MDCK细胞培养密度可以达到4.5×106cells/mL。在MOI为0.05接种流感病毒,胰酶质量浓度4μg/mL,流感病毒在MDCK细胞上可获得较高的滴度。结论 MDCK细胞用微载体培养可以达到较高的细胞密度,可以作为规模化生产新型流感病毒疫苗的主要细胞基质进行进一步的研究。     

流加微载体半连续培养分泌HBsAg的rCHO细胞过程研究

本文在固定浓度微载体半连续培养rCHO细胞动力学研究的基础上,通过逐步补加新的微载体以不断提高供细胞生长的表面,提高了细胞密度和产物的表达量。建立了流加微载体半连续培养rCHO肿细胞收获HBsAg的工艺,确定了此种培养方式的最大微载体浓度,为建立微载体连续培养rCH0细胞生产HBsAg技术,实现细胞高密度和产物高表达奠定了基础。     

用环境扫描电子显微镜研究Vero细胞在微载体上的生长

使用环境扫描电子显微镜(ESEM), 通过观察Vero细胞在Cytodex-3微载体表面上贴附铺展及增殖生长过程中的形态变化, 分析了细胞在微载体上的贴附与铺展的过程和行为; 考察了外源的层/纤粘连蛋白对细胞铺展形貌的影响, 发现细胞在微载体上的增殖生长以细胞群落延伸扩展的形式进行. 用ESEM图像计数法和结晶紫细胞核计数法获得细胞生长过程的细胞密度变化和生长曲线, 研究了接种密度对细胞生长过程的影响, 随着接种密度的增大, 细胞延迟期缩短, 表观生长速率增大, 细胞长满微载体表面所用的时间缩短. 在实验的血清浓度范围内(5%~10%), 血清浓度影响细胞的贴附, 随着血清浓度的增大, 细胞的贴附率增加, 从而影响整个生长过程.     

Vero细胞在不同微载体固定化培养中的生长和代谢

目的:比较Vero细胞在不同的商品化微载体中固定化培养的生长和代谢。方法:以Vero细胞在含1%新生牛血清的DMEM/F12中培养的细胞形态、活细胞密度和细胞活力为指标,考察Vero细胞在2D MicroHex、Biosilon、Cytodex1和Cytopore1微载体固定化培养的细胞生长;以葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、谷氨酰胺比消耗速率(qgln)和谷氨酸比生产速率(qglu)为指标,考察Vero细胞在不同微载体固定化培养的细胞代谢。结果:Vero细胞在2DMicroHex、Biosilon、Cytodex1和Cytopore1微载体固定化培养7d的活细胞密度分别为18.4×105cells/ml、21.9×105cells/ml、23.9×105cells/ml和16.2×105cells/ml,生长在Cytodex1表面的Vero细胞分布均匀、形态清晰;Vero细胞在不同微载体固定化培养的代谢指标基本相同。结论:Vero细胞在Cytodex1微载体固定化培养的效果优于其它商品化微载体,可作为目前用于病毒疫苗生产的Vero细胞固定化培养的首选微载体。     

细胞密度和营养供给对H1N1流感病毒产率的影响

探究细胞培养生产流感病毒过程中,高细胞密度引起低单位细胞病毒产率(Svy)的原因及找到解决方法。通过增加微载体浓度以及换液操作获得较高的细胞密度;考察了感染时细胞密度(CCI)和病毒感染用维持培养基对病毒产量和单位细胞病毒产率的影响。在12.6 g/L微载体培养过程中,通过换液操作,可获得高细胞密度,达到1.47×107 cells/m L。在CCI为1×107cells/m L条件时,选择合适的维持培养基,其Svy最高达到5.14×103 virions/cell,比同等条件下用DMEM维持培养基的Svy值提高了近一倍。高密度培养MDCK细胞生产流感病毒时,充分考虑不同阶段的培养条件和营养需求,可以提高细胞密度和单位细胞病毒产率,进而提高流感病毒的产量。该研究结果为工业化生产流感病毒疫苗提供了基础数据。     

微载体灌注培养制备Vero细胞狂犬病疫苗

本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术。在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6~7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0~8.5logLD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到《中国生物制品规程》2000年版要求。实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行。     

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。     

牛肾细胞在两种不同载体中培养效果的比较

目的通过牛肾细胞在两种不同载体中培养效果的比较,为牛肾细胞在细胞工厂中规模化生产提供真实的、有力的支持。方法不同代次牛肾细胞在两种载体中经过相同培养条件进行培养。结果实验中原代牛肾细胞在细胞工厂接种密度为5.5×104/cm2左右,在15 L转瓶接种密度为9.0×104/cm2左右。一代牛肾细胞在细胞工厂接种密度为6.5×104/cm2左右,在15 L转瓶接种密度为10×104/cm2左右。二代牛肾细胞在细胞工厂接种密度为7.0×104/cm2左右,在15 L转瓶接种密度为14×104/cm2左右。两种载体中牛肾细胞生长状况均能达到培养要求。结论细胞工厂能在有限的空间内利用最大限度的培养表面培养牛肾细胞,不仅节约了传代前的细胞用量,而且提高了培养后的细胞产量。     

A microcarrier-based cell culture process for the production of a bovine respiratory syncytial virus vaccine

Veterinary viral vaccines generally comprise either attenuated or chemically inactivated viruses which have been propagated on mammalian cell substrates or specific pathogen free (SPF) eggs. New generation vaccines include chemically inactivated virally-infected whole cell vaccines. The NM57 cell line is a bovine nasal turbinate persistently infected (non-lytic infection) with a strain of the respiratory syncytial virus (RSV). The potential of microcarrier technology for the cultivation in bioreactors of this anchorage dependent cell line for RSV vaccine production has been investigated. Both Cytodex 3 and Cultispher S microcarriers proved most suitable from a selection of microcarriers as growth substrates for this NM57 cell line. Maximum cell densities of 4.12×105 cells ml-1and 5.52×105 cells ml-1 respectively were obtained using Cytodex 3 (3 g l-1) and and Cultispher S (1 g l-1) in 5 l bioreactor cultures. The fact that cell growth was less sensitive to agitation rate when cultured on Cultispher S microcarriers, and that cells were efficiently harvested from this microcarrier by an enzymatic method, suggested Cultispher S is suitable for further evaluation at larger bioreactor scales (>5 l) than that described here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier cultures

An assay for measuring the number of adherent cells on microcarriers that is independent from dilution errors in sample preparation was used to investigate attachment dynamics and cell growth. It could be shown that the recovery of seeded cells is a function of the specific rates of cell attachment and cell death, and finally a function of the initial cell‐to‐bead ratio. An unstructured, segregated population balance model was developed that considers individual classes of microcarriers covered by 1–220 cells/bead. The model describes the distribution of initially attached cells and their growth in a microcarrier system. The model distinguishes between subpopulations of dividing and nondividing cells and describes in a detailed way cell attachment, cell growth, density‐dependent growth inhibition, and basic metabolism of Madin‐Darby canine kidney cells used in influenza vaccine manufacturing. To obtain a model approach that is suitable for process control applications, a reduced growth model without cell subpopulations, but with a formulation of the specific cell growth rate as a function of the initial cell distribution on microcarriers after seeding was developed. With both model approaches, the fraction of growth‐inhibited cells could be predicted. Simulation results of two cultivations with a different number of initially seeded cells showed that the growth kinetics of adherent cells at the given cultivation conditions is mainly determined by the range of disparity in the initial distribution of cells on microcarriers after attachment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A novel serum-free medium for the cultivation of Vero cells on microcarriers

Summary A novel serum-free medium for the cultivation of Vero cells on microcarriers was developed,which composed of the 1:1 mixture of Dubecco's Modified Eagle Medium: Nutrient Mixture F12, bovine serum albumin(BSA) or human serum albumin(HSA), epidermal growth factor(EGF), gelatin and Dbiotin. Both BSA and EGF were effective on cell growth, adhesion and spreading. Further addition of gelatin and biotin led to the enhanced cell adhesion and spreading without growth promoting activity. The serum-free medium was suitable for the cultivation of vero cells on several different microcarriers with cell density reached over 3×l0⁶cells/ml.     

Kinetics of prourokinase production by human kidney cells in culture

The kinetics of prourokinase production by human kidney cell line TCL were investigated using microcarriers, roller bottles and a ceramic system. The type of microcarriers used for cell cultivation affects not only growth kinetics but also the duration of post-confluent production period. Different clones of cells derived from the same line can also exhibit different growth kinetics on different types of microcarriers. The effect of serum concentration on the prourokinase production was examined. Using a low serum concentration of 1 % the production of pro-urokinase on microcarriers, roller bottles and a ceramic system was compared. In all three systems the production was sustained over an extended period reaching beyond confluence.     

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Summary Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x10⁵ cells/ml versus 7x10⁵ cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x10⁶ cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.     

微载体高密度培养Vero细胞的研究

微载体是动物细胞高密度培养的有效手段。首先在硅化的方瓶中对Cytodex 1、Cy-todex 3、Biosilon、Bellco Glass Microcarrier、CT-1、CT-3、MC-1、CT-28种国产和进口微载体进行了比较和筛选。确定以Biosilon作为Vero细胞高密度培养的首选微载体。用500mlWheaton搅拌瓶探索影响Vero细胞高密度培养的条件,表明50～60mg/ml的微载体浓度、1～2×10⁶/ml的细胞接种密度、适当的通气(95％O_2+5％CO₂)对该细胞的高密度培养具有重要意义。在200ml培养体积的Wheaton搅拌瓶中,微载体浓度为50～60mg/ml,细胞接种密度为9.24×10⁵/ml,搅拌速度为65～85r/min,经25d培养,Vero细胞密度可达2.34×10⁷/ml,表明50～60mg/ml的微载体浓度对培养细胞没有毒性。接着在1.5L CelliGen生物反应器中进行培养,细胞接种密度为4.98×10⁵/ml,培养体积为1.2L,日灌流量从0.20L逐渐加大到3.65L,经22d连接灌流培养,最终细胞密度可达2.05×10⁷/ml。     

Cultivation of mammalian cells on macroporous microcarriers.

Adherent cells can be cultivated in a stirred-tank bioreactor by attaching to microcarriers. Macroporous microcarriers, with their intraparticle space and surface area for cell growth, can potentially support a higher cell concentration than conventional microcarriers, which support cell growth only on the external surface. Chinese hamster ovary (CHO) cells and green monkey kidney (Vero) cells were cultivated on macroporous microcarriers, Cultispher-G. Cells attached to the microcarriers at a slow rate and grew to a high density. Thin sections of the microcarriers demonstrate that cells were initially on the exterior of the microcarriers and migrated into the interior as cell concentration increased. Vero cells cultivated on these microcarriers were successfully used for the production of vesicular stomatitis virus (VSV).     

Development of the optimal inoculation conditions for microcarrier cultures

The environmental conditions under which anchorage-dependent mammalian cells are grown are not necessarily those under which a culture should be initiated. Cell attachment is a physical process, and those factors which affect forces involved in cell attachment differ from the biological factors which affect cell growth. We have conducted an extensive experimental study to define clearly the optimal environmental conditions for MRC-5 cell attachment onto microcarriers. These inoculation conditions are particularly important when the serial propagation of mammalian cells on microcarriers is considered as in a human vaccine production process. The conditions which were investigated are: initial serum content (% v/v), initial pH, inoculation level (cells/bead), agitation rate (rpm), and the concentration of microcarriers (g/L). The initial distribution of attached cells was found to have a significant affect on the overall efficiency of anchorage-dependent cell cultures, and was used to evaluate attachment efficiency. Based on the experimental results, we propose an optimized protocol for the inoculation of microcarrier cultures.     

Biologicals produced from animal cells in culture--an overview

A wide spectrum of biologicals are produced from animal cells in culture. Among these biologicals are viral vaccines (human and veterinary), monoclonal antibodies, immunoregulators, enzymes, hormones, polypeptide growth factors, viral bioinsecticides, tumor antigens, cell mass as a product and reconstitution of living skin. For research and development (R&D) and production of these products requires knowledge and experience in one or more of the following advanced technologies: 1) Development of novel as well as modification of conventional cultivation equipment; 2) Adaptation of cultivation techniques to the production of the desired product; 3) Hybridization technologies; 4) Genetic engineering techniques suitable for animal cells; 5) Development of a wide range of microcarriers and fixed-bed culturing systems; 6) Microencapsulation techniques; 7) Development of suitable media for cell cultivation; 8) Adaptation of suitable protein concentration and purification techniques.