MRC-5人二倍体细胞培养条件的优化

目的对MRC-5人二倍体细胞培养条件的优化比较。方法用3种不同的培养基将MRC-5人二倍体细胞在T25方瓶和Spinner培养系统Cytodex1微载体2种培养体系进行培养比较,每天观察细胞形态,进行细胞计数,绘制生长曲线,并检测葡萄糖-乳酸值,筛选出1种较适宜培养基,用不同级别的牛血清比较其生长增殖情况。结果 3种培养基在细胞形态,细胞的贴壁、分裂以及维持等方面无明显差异。但在增殖上,在2种培养体系中MEM(43.25±0.60)×104cells/m L,(12.98±1.27)×105cells/m L与M199(35.40±1.41)×104cells/m L,(10.76±1.31)×105cells/m L及DMEM/F12(36.75±1.59)×104cells/m L,(11.22±1.42)×105cells/m L比较,差异均有统计学意义(P0.01),MEM培养液细胞增殖5.17和6.49倍,优于M199和DMEM/F12培养液。进口胎牛血清细胞增殖(4.55±0.51)×105cells/m L,优于其他3种牛血清的(4.12±1.03)×105cells/m L、(3.59±0.48)×105cells/m L和(3.53±0.52)×105cells/m L。结论 3种培养基都可以用于MRC-5人二倍体细胞的繁殖培养,但MEM培养液更佳。进口胎牛血清较其他血清有优势。     

鱼藤素对小鼠小胶质细胞 BV-2影响的实验研究

目的：研究放射增敏剂鱼藤素对小鼠小胶质细胞BV-2的影响。方法 BV-2细胞以5×104／mL密度接种到细胞培养板中,加入8个不同浓度鱼藤素干预48 h后测定细胞增殖活力。在加入1×10-6 mol／L鱼藤素后6个时间点上测定细胞增殖活力。1×10-8 mol／L鱼藤素干预48 h,测定上清液中NO水平。结果5×104／mL BV-2细胞以含10％胎牛血清DMEM培养基培养,第48 h达到细胞生长曲线的峰值。在0～1×10-5 mol／L剂量范围内,1×10-6、1×10-5 mol／L鱼藤素干预48 h后BV-2细胞的增殖活力显著降低;1×10-6 mol／L鱼藤素加入后第24、48、72 h等3个时间点上BV-2细胞增殖活力明显降低。1×10-8 mol／L鱼藤素干预48 h后BV-2细胞上清液NO水平提高。结论鱼藤素在一定的浓度时激活小胶质细胞,更高浓度时将导致小胶质细胞损伤甚至死亡。     

微囊藻毒素对大鼠睾丸支持细胞凋亡相关蛋白表达的影响

目的 利用体外培养的大鼠睾丸支持细胞研究微囊藻毒素-LR(MC-LR)对细胞中凋亡相关蛋白表达水平的影响.方法 大鼠睾丸细胞分别染毒0(对照)、0.5、1、10、20 μg/ml MC-LR溶液后培养24和48 h,采用MTT法检测细胞活性.调整细胞密度为4×106～5×106/ml,分别染毒含0(对照)、1、10 μ...     

氟化钠对大鼠成骨肉瘤细胞增殖活性及碱性磷酸酶活力和骨钙素mRNA表达的影响

目的探讨氟化钠(NaF)对成骨样细胞大鼠骨肉瘤(UMR-106)细胞增殖活性及主要成骨活性标志物碱性磷酸酶(ALP)活力和骨钙素(BGP)mRNA表达的影响。方法将处于对数生长期的UMR-106细胞分别暴露于含终浓度为0(对照)、5×10、10~2、5×10~2、1×10~3、5×10~3、1×10~4、2×10~4、4×10~4、8×10~4μmol/L NaF的DMEM培养液中孵育24、48 h,采用CCK-8检测细胞活性。将处于对数生长期的UMR-106细胞分别暴露于含终浓度为0(对照)、1×10~3、2×10~3、4×10~3μmol/L NaF的DMEM培养液(不含FBS)继续培养12、24、48 h,测定细胞培养液和细胞ALP的活力及细胞中BGP mRNA的表达水平。结果与对照组比较,1×10~3~8×10~4μmol/L NaF染毒24 h和5×10~8×10~4μmol/L NaF染毒48 h时大鼠UMR-106细胞的存活率均较低,差异有统计学意义(P0.05)。5×10、1×10~3、5×10~3、1×10~4、2×10~4μmol/L NaF染毒48 h时大鼠UMR-106细胞的存活率均低于染毒24 h时,差异有统计学意义(P0.05)。各浓度NaF染毒组UMR-106细胞培养液和细胞中ALP活力均较对照组高,除1×10~3μmol/L NaF染毒24 h的细胞培养液和1×103μmol/L NaF染毒48 h的细胞内ALP活力外,差异有统计学意义(P0.05)。随着NaF染毒浓度的升高,UMR-106细胞培养液和细胞中ALP活力均呈上升趋势;随着NaF染毒时间的延长,细胞培养液中ALP的活力呈上升趋势,而细胞中ALP的活力呈先升高后下降的趋势。2×103、4×103μmol/L NaF染毒组UMR-106细胞的BGP mRNA表达水平均较对照组高,差异有统计学意义(P0.05)。随着NaF染毒浓度的升高,UMR-106细胞BGP mRNA的表达水平呈上升趋势;随着NaF染毒时间的延长,UMR-106细胞BGP mRNA的表达水平呈下降趋势。结论在本实验浓度下,NaF对UMR-106细胞的增殖呈抑制作用,但可促进其成骨活性标志物ALP活力及BGP mRNA的表达。     

久效磷致大鼠皮肤成纤维细胞凋亡及氧化损伤的作用

目的 研究久效磷对体外培养的大鼠皮肤成纤维细胞凋亡及氧化损伤作用的影响。方法 取4只3日龄清洁级SD大鼠的背部皮肤,以组织块法培养大鼠皮肤成纤维细胞,正常传代。取第4代成纤维细胞,调整细胞密度为1.0×106/瓶,当细胞至亚融合状态,分别加入0（对照）、0.01、0.1、1μg/L的久效磷溶液培养6、12、24 h。采...     

狗肾细胞培养条件优化

目的探讨狗肾细胞培养影响因素,优化狗肾细胞培养条件。方法将狗肾细胞以1×104的密度接种到96孔板上,改变狗肾细胞的培养条件:不同的温度(35℃、36℃、37℃)、不同的培养基(MEM、DMEM、1640)、不同p H值(p H=6.4、6.7、7.0、7.2、7.5)、不同浓度的牛血清(0%、2.5%、5%、10%、15%)、不同浓度的L-谷氨酰胺(0、1、2、4 mmol/L)、不同浓度的4-羟乙基哌嗪乙磺酸(0、50、100、150 mmol/L)、不同浓度的CO2(0%、5%),每日观察细胞形态变化,并在24、48、72、96、120、144 h加入CCK-8测450 mm下的OD值,用OD值的倍数绘制MDCK细胞的增长曲线,比较不同生长条件对MDCK细胞增殖的影响。结果狗肾细胞的最优培养条件为:37℃、5%浓度的CO2、DMEM培养基、p H值6.7~7.2、5%~10%胎牛血清、4 mmol/L-谷氨酰胺、50~100 mmol/L的4-羟乙基哌嗪乙磺酸。结论可以根据工作需要,调节牛血清浓度和温度高低,来加速或减慢MDCK细胞的增殖速度。     

流感病毒在狗肾上皮细胞上的最优培养条件

目的优化不同亚型流感病毒在狗肾上皮细胞(MDCK)上最优培养条件,提高不同亚型流感病毒在MDCK细胞上的分离效果。方法将甲型H1N1、H3N2、Victoria、Yamagata四种亚型的流感病毒以不同的培养条件:不同培养温度、不同培养基、不同的牛血清浓度、不同L-1(对-甲苯磺酰)苯丙胺酰氯甲酮(TPCK)胰酶浓度、不同的接种量、不同的接种时间接种到MDCK细胞上,在24h、48h、72h、96h取培养物上清测血凝滴度,比较不同亚型流感病毒在MDCK细胞病变速度和病毒含量的差异。结果甲型H1N1流感病毒的最优培养温度为35℃,DMEM培养基,无牛血清,TPCK胰酶浓度为1-3μg/ml,接种300ml,不吸附或吸附1.5-3.0小时;H3N2病毒的最优培养温度为34℃,DMEM培养基,无牛血清,TPCK胰酶浓度为1μg/ml,吸附1.5小时,接种300ml;Victoria流感病毒的最优培养温度为35℃,DMEM培养基,无牛血清,TPCK胰酶浓度为0.5-2.0μg/ml,接种200ml,吸附1.5小时;Yamagata流感病毒的最优培养温度为35℃,DMEM培养基,无牛血清,TPCK胰酶浓度为1μg/ml,接种200ml,吸附1.5小时。结论根据流感病毒不同亚型来选择流感病毒的培养条件可获得更好的实验结果。     

两种方法处理支原体污染的疗效观察

支原体是细菌培养中最常见、不易被察觉又很能干扰实验结果的一种污染。从1952～1972年间,有人检查了54个实验室的 9700个各类培养细菌从中发现有11％的细胞受到支原体污染[1]。目前发现支原体已有数十种之多。抗生素对支原体污染不能奏效,根据支原体对热耐受性差的特点,本实验拟用加温和药物处理体外培养被污染的细胞,以杀灭支原体。1 材料与方法1.1 药品试剂　DMEM培养基为Gibco公司产品;胰蛋白酶、Hoechst33342和碘化丙啶（PI）荧光染料均为Sigma公司产品;胎牛血清为血液学研究所生产;土槿甲酸由本院药学教研室提供。1.2 细胞来源与培养 人胃低分化粘液腺癌细胞系（MGC80-3）由北京医科大学肿瘤研究所提供,体外培养于DMEM培养基中,内含10％胎牛血清;2mmol/L谷氨酰胺;100μ/ml青霉素、100μg/ml链霉素,置5％CO2孵育箱中37℃培养。1.3 加温处理被污染的细胞 将支原体污染的细胞按5×104个/ml密度接种于25ml玻璃培养瓶内,待接种后48h实验组细胞     

HBV阳性血清体外感染HepG-2细胞方法建立

目的建立乙型肝炎病毒(HBV)阳性血清体外感染人肝癌细胞系HepG-2的方法。方法低温同步化处理HepG-2细胞后用高浓度HBV阳性血清与含有4%聚乙二醇的无血清伊格尔极限必需培养基(MEM)共孵育HepG-2细胞,设阴性对照组和空白对照组;18 h后加入含10%胎牛血清的MEM继续培养6 d,每隔24 h收集细胞和培养上清,荧光定量PCR检测HBV DNA,间接免疫荧光技术检测细胞内乙型肝炎病毒表面抗原(HBsAg)。结果 HBV阳性血清感染组HepG-2细胞内和培养上清中在感染后第1 d可检测到HBV DNA,分别为(16.04±7.99)×103、(8.84±3.97)×103 copies/mL,细胞内DNA含量在第2 d达到(3.51±1.86)×105 copies/mL,然后逐渐下降,在第4 d降到检测下限以下,而培养上清中病毒DNA在检测的时间内逐渐升高,在第6天达到(8.41±5.34)×105 cop-ies/mL;间接免疫荧光检测感染组细胞膜和胞浆中均有HBsAg表达;传代培养感染细胞后,在第1代细胞培养上清和细胞内均可检测到HBV DNA(3.58×105、7.34×105 copies/mL),第2代培养上清中可检测到少量HBV DNA(2.89×103 copies/mL),但细胞内检测到HBV DNA低于检测下限(896 copies/mL),第3代细胞的上清和细胞内均未检测到HBV DNA。结论 HBV阳性血清在一定条件下可以感染HepG-2细胞,病毒能在细胞内进行短期复制。     

铁皮石斛对高糖环境下人脐静脉内皮细胞线粒体膜电位的影响

目的研究铁皮石斛多糖对体外高糖环境下人脐静脉内皮细胞线粒体膜电位的影响及其作用机制。方法将常规培养的人脐静脉内皮细胞分为DMEM/低糖培养基组(对照组)、DMEM/高糖培养基组(33.3 mmol/L)(高糖组)、3个不同浓度铁皮石斛多糖高糖组(100、200、400μg/ml)共5组,培养48 h后MTT法检测细胞活力,流式细胞仪检测线粒体膜电位。结果 33.3 mmol/L高糖环境下人脐静脉内皮细胞生长受抑,细胞线粒体膜电位降低,铁皮石斛多糖能剂量依赖性地有效拮抗上述改变,且有统计学意义(P0.05)。结论铁皮石斛多糖可能通过升高高糖环境下的人脐静脉内皮细胞线粒体膜电位,增加高细胞活力,而对其具有较好的保护作用。     

Establishment of a mink enteritis vaccine production process in stirred-tank reactor and Wave Bioreactor microcarrier culture in 1-10 L scale

A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave Bioreactors. Transfer from a roller bottle-based production process into large-scale microcarrier culture with starting concentrations of 2g/L Cytodex 1 microcarriers and 2.0 x 10(5)cells/mL was successful. A maximum cell yield of 1.2 x 10(6)cells/mL was obtained in stirred-tank microcarrier batch culture while cell numbers in the Wave Bioreactor could not be determined accurately due to the fast sedimentation of microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus-host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave Bioreactor using a multiple harvest strategy. Maximum virus titres of 10(6.6) to 10(6.8) TCID(50)/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.     

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.     

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.     

Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media

A process for equine influenza virus vaccine production using a microcarrier system (Cytodex 1) in a 2 L Wave bioreactor is described. Growth of Madin Darby canine kidney (MDCK) cells in serum containing GMEM medium (SC) is compared to growth in serum-free Ex-Cell MDCK medium (SF) without washing steps and medium exchange before infection. Cultivations with microcarrier concentrations of 2 and 4 g/L for both media are shown. Metabolic data from carbon and amino acid metabolism are discussed. Additionally, in roller bottle experiments the influence of multiplicity of infection (moi) and trypsin concentration on the HA value was investigated. Analysis of HA and TCID(50) at 37 degrees C showed a stable HA of maximum 2.6 log HA/100 microL for 2 weeks. Peak TCID(50) titers of 10(7.7) viruses/mL were achieved 20h post infection, but infectivity was below detection limit after 150 h. Cell attachment onto microcarriers under serum-free conditions was improved by Ca(2+) addition and by cell harvesting without trypsin using only an EDTA/PBS solution. For the wave cultivation maximum virus titers of 2.3-2.6 log HA units/100 microL were reached from infection with a moi of 0.05. However, in SF medium pH dropped to less than pH 6.8 which resulted in lower HA titers of 1.7 log HA units/100 microL. For the higher microcarrier concentration (4 g/L) medium exchange steps (500 mL) were needed for both media. Omission of the washing step and medium exchange before infection in SF medium clearly simplified the influenza production process; however, for higher virus yields a better pH control of the wave bioreactor would be required. Higher cell densities (2.8 x 10(6) cells/mL for 2 g/L microcarrier) and better attachment compared to stirred tank bioreactors showed, that the wave bioreactor is a good alternative to stirred tank processes for expanding production capacities in case of a pandemic.     

两株人胚胎干细胞的无饲养层扩增

目的:建立两株中国人胚胎干细胞(hESCs)的无饲养层的培养体系,观察条件培养基所需的饲养层密度,并探索最佳的传代方式。方法:两株人胚胎干细胞hES-846XX和hES-1846XY分别使用不同密度(3×105/mL、6×105/mL和1.2×106/mL)获得的条件培养基(CM)在无饲养层培养体系培养12代以上,用机械分离法和Ⅳ型胶原酶法进行传代,观察比较结果并对所得hESCs鉴定。结果:6×105/mL～1.2×106/mL之间的密度都是合适密度;Ⅳ型胶原酶在长期传代中优于机械法传代;所得细胞维持干细胞未分化状态和全能性。结论:无饲养层的培养体系可以用于中国胚胎干细胞培养,两株中国人胚胎干细胞(hESCs)的倍增周期、所需饲养层密度及分化率等方面与国外hESCs有明显差别。     

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.     

Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture

A complete serum-free process without washing steps and medium exchange before infection for influenza A virus vaccine production (equine and human) is described for cultivation in roller bottles and in a 5-L stirred tank microcarrier system. Adherent Madin-Darby canine kidney cells (MDCK) were adapted from growth in serum containing GMEM medium to growth in serum-free Ex-Cell MDCK medium. Roller bottle experiments showed that the medium exchange step, typically required for serum containing vaccine production processes, could be omitted without losses in virus titre and without limitations in glucose or glutamine supply in the cultivation medium. The serum-free medium could even be used glutamine-free as it contained pyruvate, resulting in very low levels of ammonia. Cell attachment onto microcarriers was critical. Therefore, microcarriers had to be preconditioned in medium. Also, trypsin concentration used for inoculum preparation had to be reduced. After these modifications 1.3 x 10(6)cells/mL were obtained after 97 h (2g/L Cytodex 1) of cell growth. Maximum virus titres of 2.3-2.9 log HA units/100 microL were obtained from infections with a multiplicity of infection (moi) of 0.05-0.10 for human and equine influenza A virus. Metabolite and amino acid profiles as well as on-line data for the serum-free process are compared with the serum containing process. Omission of the medium exchange before infection clearly simplified the process and reduced sterility risks due to washing steps.     

Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI 1640 medium without animal protein

Madin Darby canine kidney (MDCK) cells were adapted to serum-free RPMI 1640 medium and used for cultivation of canine viruses. RPMI 1640 medium was supplemented with a soybean peptone, L-glutamine and antibiotics, so that the protein concentration was less than 5 microg/ml (RPMI/SP medium). The resulting adapted MDCK-SP cells showed steady growth after the twenty-eighth passage in RPMI/SP medium (MDCK-SP cell culture). Canine distemper virus, canine parvovirus, canine adenoviruses and canine parainfluenza virus, which are the principal components of canine combined virus vaccines, grew in the MDCK-SP cell culture as efficiently as the parental MDCK cells cultured in the conventional Eagle's MEM containing fetal bovine serum. Consequently, the use of MDCK-SP cell culture can make current canine vaccine products much safer, of higher quality and at lower cost.     

Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells

Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected and the PI-3 virus production in 12L attained 12 log(10) TCID(50). Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors.