Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Embryonic stem (ES) cells have the ability to differentiate in vitro into a wide variety of cell types with potential applications for tissue regeneration. However, a large number of cells are required, thus strengthening the need to develop large-scale systems using chemically defined media for ES cell production and/or controlled differentiation. In the present studies, a stirred culture system (i.e. spinner flask) was used to scale-up mouse ES (mES) cell expansion in serum-containing (DMEM/FBS) or serum-free medium, both supplemented with leukemia inhibitory factor (LIF), using either Cytodex 3 or Cultispher S microcarriers. After 8 days, maximal cell densities achieved were (1.9+/-0.1), (2.6+/-0.7) and 3.5x10(6)cells/mL for Cytodex 3 in DMEM/FBS, Cultispher S in DMEM/FBS and Cultispher S in serum-free cultures, respectively, with fold increases of 38+/-2, 50+/-15 and 70. Both microcarriers were suitable to sustain mES cell expansion, though the macroporous Cultispher S seemed to be advantageous in providing a more protective environment against shear stress forces, which harmful effects are exacerbated in serum-free conditions. Importantly, mES cells expanded under stirred conditions using serum-free medium retained their pluripotency and the ability to commit to the neural lineage.     

Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.     

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.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

A novel process for the production of a veterinary rabies vaccine in BHK-21 cells grown on microcarriers in a 20-l bioreactor

We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.     

Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture

In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.     

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 °C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.     

Microcarrier culture of fish cells and viruses in cell culture bioreactor.

This article deals with the culture of grass carp (Ctenopharyngodon idellus) lip and embryo cells on Cytodex 3 and GT-2 microcarriers in a 1.5-L cell culture bioreactor to propagate grass carp hemorrhage virus. The cells and viruses were successfully cultivated at 26 degrees C, pH 7.0, and dissolved oxygen 40% of air saturation. The cell density achieved was as high as 7.4 x 10(6) cells/mL, and the virus titre reached 6.75 log LD50/0.5 mL from an initial 3.00 log LD50/0.5 mL. The results present broad prospects for fish virus vaccine production.     

应用细胞工厂建立人用狂犬病疫苗连续培养多次收获工艺

目的为提高生产效率、增加原代地鼠肾细胞单产量及狂犬病病毒产量,建立人用狂犬病疫苗（地鼠肾细胞）连续培养多次收获工艺。方法选用12-14日龄SPF地鼠,无菌取肾经消化,制备成细胞悬液,分装到40层细胞工厂并培养细胞成单层;接种狂犬病病毒固定毒aG株,连续培养病毒并多次收获。分别对同一细胞批制备的多个单次病毒收获液的免疫原性、病毒滴度和地鼠肾细胞蛋白质含量进行检测。结果用40层细胞工厂培养原代地鼠肾细胞和狂犬病病毒,细胞接种浓度为1．0×10。～1．5x10。cells／mL,（36±1）℃培养72h成致密单层;按0．1MOI病毒接种,可进行6次收获病毒;多个单次病毒收获液病毒滴度均不低于6．0lgLD50／mL;免疫原性检查保护指数不低于100;地鼠肾细胞蛋白质残留量随着收获次数的增加而不断降低。结论用细胞工厂建立了人用狂犬病疫苗连续培养多次收获工艺,能显著提高地鼠。肾单产量,增加产能。     

Production and glycosylation of recombinant beta-interferon in suspension and cytopore microcarrier cultures of CHO cells

Microcarriers are suitable for high-density cultures of cells requiring surface attachment and also offer the advantage of easy media removal for product recovery. We have used the macroporous microcarriers Cytopore 1 and 2 for the growth of CHO cells producing recombinant human beta-interferon (beta-IFN) in stirred batch cultures. Although these cells may grow in suspension, in the presence of Cytopore microcarriers they become entrapped in the inner bead matrix where they can be maintained at high densities. Cell growth rates were reduced in microcarrier cultures compared to suspension cultures. However, the beta-IFN yield was up to 3-fold greater as a result of an almost 5-fold higher specific productivity. Maximum productivity was found in cultures containing 1.0 mg/mL of Cytopore 1 or 0.5 mg/mL of Cytopore 2 with a cell/bead ratio of 1029 and 822, respectively. Beta-IFN molecules aggregated in the later stages of all cultures, causing a decrease in response by ELISA. However, the degree of aggregation was significantly less in the microcarrier cultures. The N-linked glycans from beta-IFN were isolated and analyzed by normal phase HPLC. There was no apparent difference in the profile of glycans obtained from each of the suspension and Cytopore culture systems. This suggests that Cytopore microcarriers may be useful in bioprocess development for enhanced recombinant glycoprotein production without affecting the glycosylation profile of the protein.     

Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture

It is not well understood how changes from suspension to microcarrier cultures affect cell growth, metabolism, and yield of recombinant proteins. To investigate the effects of culture conditions on cell characteristics, fed-batch bioreactor cultures were performed under different culture conditions (suspension cultures, cultures attached to Cytodex 3 and Cytopore 1 microcarriers) using two different Chinese hamster ovary cell lines producing either secreted human placental alkaline phosphatase (TR2-255) or tissue plasminogen activator (CHO 1-15-500). In controlled, agitated bioreactors, suspension cultures reached cell densities and product titers higher than those in microcarrier cultures, in contrast to the results in static flask cultures. Growth and metabolic activities showed similar trends in suspension and microcarrier culture regardless of cell line. However, the responses of the specific productivities to the different culture conditions differed significantly between the cell lines.     

食管癌细胞诱导肥大细胞迁移的小鼠模型

目的建立小鼠肥大细胞（mast cell,MC）迁移模型,探讨化疗药物三氧化二砷对人食管癌EC109细胞株生长的杀伤机理。方法利用肥大细胞的特征性蛋白酶抗体及其免疫荧光标记MC和PI标记EC109细胞内DNA;以流式细胞术分析小鼠腹腔液中肥大细胞各亚型的百分率及肿瘤细胞周期变化;使用激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒的分布。并通过组织化学方法,观察各种诱导处理后小鼠肠组织肥大细胞由肠道向腹腔移动的变化。结果①根据流式细胞仪点图分布分析,将小鼠肥大细胞分为：类胰蛋白酶阳性、类糜蛋白酶阴性（MC T）;类糜蛋白酶阳性、类胰蛋白酶阴性（MC C）和类胰蛋白酶阳性、类糜蛋白酶阳性（MC TC）三种亚型,且T型MC明显多余TC型和C型（P〈0.05）;以共聚焦显微镜显示三种亚型的MC均含有丰富的分泌颗粒并分布于细胞膜内侧,为其出芽突起形成储备的状态。②经组织切片观察到诱导处理后小鼠肠组织MC由肠道向腹腔移动,且胰酶对MC的诱导作用大于食管癌细胞和As2O3。③经诱导迁移入腹腔的MC可能与癌细胞周期由S期向G2/M期跨越相关;As2O3能延迟食管癌细胞的G0/G1期,阻碍细胞向S期跨越,从而抑制食管癌细胞的生长。结论食管癌细胞移入小鼠腹腔,主要诱导T型MC参与免疫反应。在生物机体内环境（这里指MC影响）的条件下,As2O3对肿瘤细胞生长的作用主要表现为促使癌细胞周期的G0/G1期向S期跨越延迟,或G2/M期进入细胞分裂的延迟。     

Application of a serum-free medium for the growth of Vero cells and the production of reovirus

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.     

Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium

Requirements of eliminating animal proteins from cell culture have intensified in recent years, with the pressure of regulatory agencies related to biopharmaceuticals production. In this work, the substitution of fetal bovine serum by yeastolate and a soy hydrolysate (Hy Soy) for the culture of Drosophila melanogaster Schneider 2 cells transfected for the production of rabies virus G glycoprotein was evaluated. TC100 supplemented with glucose, glutamine, lipid emulsion and Pluronic F68 was employed as basal medium. Results show that yeastolate was more efficient on cell growth stimulation than Hy Soy. Cells adapted in medium formulation supplemented with 3 g/L yeastolate, 1% lipid emulsion, 10 g/L glucose, 3.5 g/L glutamine and 0.1% Pluronic F68 attained a maximum concentration of 10.7 x 10(6) cells/mL, with the expression of 9.4 ng/mL G glycoprotein.     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

Inactivated Rabies Vaccine Produced from the Flury LEP Strain of Virus Grown in BHK-21 Suspension Cells

Suspension cultures of BHK-21 cells maintained at 32 to 33 C were infected with the Flury LEP strain of rabies virus. By using a cell concentration of 2.0 x 10(6) to 2.5 x 10(6) cells per ml infected at a multiplicity of 0.05, high titers of extracellular virus were reached in 96 to 120 h, and potent inactivated vaccines were prepared from culture fluids harvested between 96 to 168 h. The addition of 1% bovine serum to the maintenance medium resulted in an increase in virus yields and vaccine potency. Estimation of the number of infected cells by immunofluorescent procedures proved a rapid and reliable guide to the virus content of suspension cultures.     

Insights into adenoviral vector production kinetics in acoustic filter-based perfusion cultures

One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.     

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-beta1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF-beta1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation.     

Hepatocyte viability and protein expression within hydrogel microstructures

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.     

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.