Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

中国森林生态系统N平衡现状

由于N饱和生态系统的出现,森林生态系统作为环境污染储蓄库的认识受到挑战。收集了近十余年来全国各地森林N素循环的研究资料,通过对目前大气N沉降、森林生物固N、森林生态系统N的流失、淋失、挥发等各项收支参数的分析,借助农田养分收支平衡的估算思路和方法对全国森林生态系统N平衡进行了估算。结果表明,我国森林生态系统N的输入大于输出,全国森林生态系统年容纳大气N约为736万t,其中约176万t来自于大气N沉降,约599万t来自于生物固N。而进入到森林生态系统中的N约16万t固定在木材中用以维持森林蓄积的增加,其余绝大部分则保存于森林土壤,使得森林土壤全N含量大约以每年0.002%的速率增长。但不断增加的N素输入并未导致森林生态系统N饱和,全国的森林蓄积仍保持增长的趋势,森林生态系统在N的生物地球化学循环过程中起着重要的调节作用,仍是环境N的储蓄库,对于调节气候,防治污染具有重大作用。     

Development of an animal‐component free medium for vero cells culture

This work describes the development of an animal‐component free medium (IPT‐AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum‐free medium (SFM) referred as IPT‐SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT‐SFM was further improved to obtain an animal‐component free medium named IPT‐AFM. IPT‐AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue‐culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non‐animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT‐AFM were investigated in T‐flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 ± 0.18 and a specific growth rate μ 0.019 ± 0.003 h^?1 were achieved in IPT‐AFM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The role of vitamins and amino acids on hybridoma growth and monoclonal antibody production

A balanced supplementation method was applied to develop a serum and protein- free medium supporting hybridoma cell batch culture. The aim was to improve systematically the initial formulation of the medium to prevent limitations due to unbalanced concentrations of vitamins and amino acids. In a first step, supplementation of the basal formulation with 13 amino acids, led to an increase of the specific IgA production rate from 0.60 to 1.07 pg cell⁻¹ h⁻¹. The specific growth rate remained unchanged, but the supplementation enabled maintenance of high cell viability during the stationary phase of batch cultures for some 70 h. Since IgA production was not growth- related, this resulted in an approximately4-fold increase in the final IgA concentration, from 26.6 to 100.2 mgl⁻¹. In a second step, the liposoluble vitamins E and K₃ were added to the medium formulation. Although this induced a slightly higher maximal cell concentration, it was followed by a sharp decline phase with the specific IgA production rate falling to 0.47 pg cell⁻¹ h⁻¹. However, by applying a second cycle of balanced supplementation with amino acids this decline phase could be reduced and a high cell viability maintained for over 300 h of culture. In this vitamin- and amino acid- supplemented medium, the specific IgA production rate reached a value of 1.10 pg cell⁻¹h⁻¹ with a final IgA concentration of 129.8 mgl⁻¹. The latter represents an increase of approximately5-fold compared to the non- supplemented basal medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of roller culturing of influenza virus vaccine strains in MDCK and Vero cell cultures using plant origin components

Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml).     

Establishment and characterization of a porcine kidney cell line, FS-L3, which forms unique multicellular domes in serum-free culture

Summary A stable porcine kidney epithelial cell line, FS-L3, was established and maintained in Eagle’s minimum essential medium containing 0.295% tryptose phosphate broth, 0.5% Bacto Peptone, and 10 mM N, N-Bis (2-hydroxyethyl)-2-aminoethanesulfonic acid without any serum. The mode of chromosomes is 37 to 38. The FS-L3 cells formed fluid-filled, multicellular, three-dimensional domes on a single monolayer. The number of domes increased markedly after further cultivation. The origin of this cell line was confirmed as porcine by hybridization using PRE-1, which can be detected as a specific sequence in the porcine genome. It was also found that FS-L3 cells were free from possible adventitious viruses and mycoplasmas.     

The origin and characteristics of a pig kidney cell strain,LLC-PK1

Summary A stable epithelial-like pig kidney cell strain has been established. This strain has been carried through more than 300 serial passages, has remained free of microbial and viral contaminants, and has retained a near diploid number of chromosomes. Attempts to produce tumors with these cells in immunosuppressed laboratory animals have been uniformly negative. The cells have grown rapidly in monolayer cultures with a split ratio of 1 to 15 at weekly intervals, but have failed to proliferate in suspension cultures. A subline adapted to growth on serum-free medium 199 has been carried through 145 passages on this medium. Several unusual morphologic features have been observed in these cultures including three-dimensional “domelike” structures. These cells have been found susceptible to some viruses and have been especially useful for viruses of domestic animals. LLC-PK₁ cells have produced significant levels of plasminogen activator. Histopathological examinations of animal tissues were done by Dr. C. G. Culbertson. Tests for mycoplasma were performed by Mr. D. H. Holmes.     

Antiviral activity of oxidized polyamines

Summary. Polyamines, oxidized by serum amine oxidase, yield aminoaldehydes and hydrogen peroxide. Acrolein may be formed from the aminoaldehydes by a spontaneous β-elimination process. These oxidation products “oxidized polyamines” inhibit bacterial growth and exhibit anticancer activity. The antimicrobial activity of oxidized polyamines is not limited to bacteria; and the inactivation of bacterial viruses, plant viruses and animal viruses, was also reported. Bacteriophages of the T-odd series are permeable and were inactivated by oxidized polyamines. The inactive phages absorb to their bacterial host and injected their DNA, which formed a stable inactive complex with the aminoaldehydes. Aminoaldehydes, synthesized chemically, also inactivated viruses. The growth of the plant viruses: Tobacco mosaic virus, Potato virus X and Alfalfa mosaic virus was also inhibited by oxidized polyamines. The animal viruses, which were inactivated by oxidized polyamines included Myxoviruses (influenza and Newcastle disease viruses), West Nile, vaccinia and Sindbis viruses. These findings may have practical implications.     

兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1

The hydrogen (H2) production potential of the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 was evaluated at 85 degrees C. In batch cultivation using a complex medium supplemented with elemental sulfur (S0), evolution of H2S and CO2 was observed in the gas phase. When S0 was omitted and pyruvate or starch was added in the medium, the cells produced H2 at high levels instead of H2S. As the level of H2 appeared to correlate with the specific growth rate, analysis in continuous cultures was performed to develop a continuous H2 production system. In a steady-state condition at a dilution rate of 0.2 h-1, a continuous H2 production rate (per gram dry weight, gdw) of 24.9 and 14.0 mmol gdw-1 h-1 was observed in media supplemented with pyruvate and starch, respectively. In both cultivations, a high accumulation of acetate and alanine was found as metabolites. When the dilution rates were elevated in the medium with pyruvate, steady-state growth was observed up to 0.8 h-1, and a maximum H2 production rate of 59.6 mmol gdw-1 h-1 was obtained. Based on the experimental results along with data of the entire genome sequence, the metabolic pathway of the strain relating to starch and pyruvate degradation is discussed.     

Bcl-2 over-expression reduced the serum dependency and improved the nutrient metabolism in a NS0 cells culture

The over-expression of Bcl-2 has greatly improved the culture period, specific growth rate, and maximum viable cell density of NS0 cells culture under low serum condition. Further analysis of these data suggests that a saturation model of the Monod type can be used to represent the relationships of specific growth rate and initial serum concentration. The μ_max andK _s for the Bcl-2 cell line is 0.927 day⁻¹ and 0.947% (v/v) respectively, which are 21% greater and 7% lower respectively than its control counterpart. Study on the amino acid supplementation revealed that Bcl-2 cell lines possess greater improvement in the specific growth rate and maximum viable cell density compared to the control cell lines. A further increase in the amino acid supplementation has resulted a 17% decrease in specific growth rate and no improvement in maximum viable cell density in the control culture. However, the Bcl-2 cell line exhibited a better growth characteristic in this culture condition compared to that of control cell lines. The higher specific growth rate and maximum viable cell density of the Bcl-2 cell line in medium fortified with serum and MEM EAA suggested a more efficient nutrient metabolism compared to that in the control cell line. The low serum and amino acid utilisation rate and the higher cell yield may prove to be important in the development of serum/protein free culture.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

Optimisation of Microbial 3-methylcatechol Production as Affected by Culture Conditions

Pseudomonas putida MC2 can be used to produce catechols. The accumulation, specific and volumetric production rates of 3-methylcatechol from toluene have been studied. Production rates were shown to depend on growth medium, pH and toluene concentration. Minimal glucose medium and rich LB medium were the best growth media for 3-methylcatechol production. A lower pH often resulted in slower growth and a higher specific production rate, but a lower volumetric production rate. Specific production rates also increased at higher initial toluene concentrations. The best process conditions in terms of substrate conversion and specific production rate were found in minimal glucose medium at an initial aqueous toluene concentration of 1.0 &#117 mM and an initial pH of 6. At pH 7 and 2.0 &#117 mM toluene, more product was accumulated at a lower specific rate, but at a higher volumetric production rate.     

Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat

The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (mu). Under aerobic growth conditions fimbriae production increased with higher mu values till mu = 0.40 h-1 and decreased again at mu values close to mu max (0.48 h-1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to mu max (0.16 h-1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of mu. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+ cell and a slower reduction in the percentage of P+ cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+ cells and a constant amount of fimbriae per P+ cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+----P- switch between two steady states was 2.7 x 10(-2). In batch culture the frequency of the P(-)----P+ switch was minimally 2.9 x 10(-2). The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.     

Time-dependent density functional theory study of the excited-state dihydrogen bonding: clusters of 2-pyridone with diethylmethylsilane and triethylgermanium

Density functional theory (DFT) was carried out to identify the existence of intermolecular dihydrogen bonds of the 2-pyridone (2PY)-diethylmethylsilane (DEMS) and 2PY-triethylgermanium (TEGH) clusters in the ground state. The H···H distances of both clusters are shorter than the sum of their van der Waals radii. Thus, intermolecular dihydrogen bonds N–H•••H–Si and N–H•••H–Ge exist in the 2PY-DEMS and 2PY-TEGH clusters, respectively. Based on the ground-state conformations, intermolecular dihydrogen bonds N–H•••H–Si and N–H•••H–Ge in the electronically excited state of the 2PY-DEMS and 2PY-TEGH clusters were also investigated using time-dependent density functional theory (TDDFT). Electronic transition of the 2PY-DEMS cluster resembles that of the 2PY-TEGH cluster. Their S₁ state is a locally excited (LE) state centered on 2PY moiety. The H•••H distances of the 2PY-DEMS and 2PY-TEGH clusters both stretch in the S₁ state compared to those in the ground state. Upon electronic excitation, intermolecular dihydrogen bonding N–H•••H–Si and N–H•••H–Ge can weaken with decreasing dihydrogen bonding energies.     

Influence of Cultivation Parameters on Growth and Microcystin Production of Microcystis aeruginosa (Cyanophyceae) Isolated from Lake Chao (China)

Microcystis aeruginosa isolated in 2005 from the shallow eutrophic Lake Chao (Anhui, China) was investigated in terms of growth parameters and microcystin production under varying nutrient concentrations (P, N) and pH values (abiotic factors) as well as under the influence of spent medium of the non-toxic cyanobacterium Synechocystis sp. (biotic factors). Stimulating effects on growth were observed at the alkaline pH value (10.5), whereas toxin production was significantly increased under phosphate-P limitation (0.6 mg L⁻¹ medium). Within a broad range of nitrate–N concentrations (41.2–247.2 mg L⁻¹ medium), no significant influence on cell growth and microcystin production was observed; however, N-starvation resulted in a typical decrease of growth and toxicity. In addition, cryopreservation of M. aeruginosa evidenced the decrease of toxin production by time-dependent exposure with the cryoprotectant dimethyl sulfoxide under thawing conditions without affecting the growth of the cyanobacterial cells.     

Cell cultures of Rauwolfia sellowii: growth and alkaloid production

Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 0.2 mg l-1 kinetin and 30 g l-1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h-1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro mass production of <Emphasis Type="Italic">Ruta gravoelens</Emphasis> L. for secondary products production

Hypocotyls explants of 1-week-old Ruta gravoelens L. seedlings showed high regeneration ability on Murashige and Skoog’s medium supplemented with 5.2 mM H₂PO₄ ⁻, 250 mM adenine sulphate, 3.0% sucrose and 8.87 μM BA. Increasing phosphate and adenine sulphate concentrations had pronounced effects on organogenesis and shoot growth compared to the stander MS medium. Multiple shoots were successfully maintained for 60 days in static liquid medium without any hyperhydricity phenomena. Uncountable shoot buds and shoots were developed on the leaf surface which was in contact with the medium. The growth index increased linearly during the culture course (R ² = 0.992–0.999) and the highest GI (30.43) was achieved on medium enriched with 90 mM nitrogen and 4.5% sucrose. Medium supplemented with 6.0% sucrose and 60 mM N yielded the highest biomass (4.37 g per flask). However, medium with the stander N content and 6.0% sucrose developed the maximum percentage of biomass (6.67%). The highest percentage of furanocoumarins (11.46 mg g⁻¹ dry weight) calculated as xanthotoxin was produced in medium enriched with 90 mM N and 4.5 or 6.0% sucrose, whereas the maximum rutin (73.5 mg g⁻¹ dry weight) was on medium supplemented with 90 mM N and 4.5% sucrose. Developing system seems to be used for secondary metabolites production because the shoot rooting did not appear. So, all energy is devoted for shoots multiplication, their growth and secondary metabolites production.     

Development of Epitope-Blocking ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses

Background

Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available.

Methodology/Principal Findings

Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274–281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization.

Conclusions/Significance

The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.     

Influence of cultural conditions on growth and lipolytic activity inNocardia asteroides

The growth and the production of extracellular and intracellular lipases were measured fromNocardia asteroides grown under different cultural conditions. Maximal growth and intracellular and extracellular activities were observed at 3 d after inoculation. Among the tested media, synthetic medium induced maximal growth and extracellular activity, whereas tryptic soy broth induced the maximal intracellular lipase activity. The best carbon and nitrogen sources for growth and lipolytic activity were glucose, fructose, glutamate and nitrate, respectively. The optimal C∶N ratio for growth was in the range of 1∶4 to 2∶3 and for lipase activity the range was 2∶3 to 3∶2. Anything above or below this range was detrimental to the organism and its enzyme activity. Under the conditions of this study,N. asteroides grew best and had the highest lipase activity when compared toN. brasiliensis andN. caviae.