Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: a biological system approaching a quenched Kardar-Parisi-Zhang universality.

The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α=0.63±0.04,ß=0.75±0.05, and z=0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model.

Development of an in situ detachment protocol of Vero cells grown on Cytodex1 microcarriers under animal component-free conditions in stirred bioreactor.

Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L(-1). Cells were also grown in spinner flask on 2 g L(-1) Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78 ± 8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70 ± 19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L(-1) Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.

The effects of confluency on cell mechanical properties.

Mechanical properties of cells depend on various external and internal factors, like substrate stiffness and surface modifications, cell ageing and disease state. Some other currently unknown factors may exist. In this study we used force spectroscopy by AFM, confocal microscopy and flow cytometry to investigate the difference between single non-confluent and confluent (in monolayer) Vero cells. In all cases the stiffness values were fitted by log-normal rather than normal distribution. Log-normal distribution was also found for an amount of cortical actin in cells by flow cytometry. Cells in the monolayer were characterized by a significantly lower (1.4-1.7 times) Young's modulus and amount of cortical actin than in either of the single non-confluent cells or cells migrating in the experimental wound. Young's modulus as a function of indentation speed followed a weak power law for all the studied cell states, while the value of the exponent was higher for cells growing in monolayer. These results show that intercellular contacts and cell motile state significantly influence the cell mechanical properties.

Expression and functional characterisation of the putative SARS coronavirus non-structural proteins X1-X5.

1. We produced mammalian expression vectors encoding the SARS coronavirus (SARS-CoV) accessory proteins with or without the fluorescence protein tag and cell lines with stable expression of these proteins. 2. The cellular localisation and function of the SARS-CoV accessory proteins was determined. 3. SARS 6 and SARS 8b proteins are localised to the endoplasmic reticulum and nucleus/cytoplasm, respectively, and both proteins stimulate host cell DNA synthesis.

Molecular and genetic characterisation of the SARS coronavirus auxiliary protein X1 in Drosophila.

1. We have generated monoclonal antibodies against the SARS coronavirus (SARS-CoV) X1/3a protein (3a), which are suitable for western blotting, immunocytochemistry, and immunohistochemistry. 2. We have established and characterised an in-vivo 3a transgenic Drosophila model, and demonstrated its usefulness in studying SARS-CoV 3a gene function. 3. We validated our in-vivo findings on 3a gene function in mammalian Vero E6 cells. 4. Our findings raise the possibility of using ion channel blockers as a novel approach to suppress SARS-CoV-induced cell death.

In vitro effects of selenium deficiency on West Nile virus replication and cytopathogenicity.

BACKGROUND: Selenium (Se) deficiency plays an important role in viral pathogenesis. To understand the effects of Se deficiency on West Nile virus (WNV) infection, we analyzed cytopathogenicity, apoptosis and viral replication kinetics, using a newly developed Se-deficient cell culture system. RESULTS: Both Vero and SK-N-SH cells grown in Se-deficient media exhibited a gradual loss of glutathione peroxidase (GPx1) activity without any significant effect on cell growth and viability. In SK-N-SH cells, Se deficiency had no effect on the expression of key antioxidant enzymes, including manganese- and copper-zinc superoxide dismutase (MnSOD and CuZnSOD), catalase and inducible nitric oxide synthase, whereas Vero cells demonstrated a significant increase in the expression of MnSOD and an overall increase in oxidative stress (OS) at day 7 post-induction of Se deficiency. At 2 days after infection with WNV, CPE and cell death were significantly higher in WNV-infected Se-deficient Vero cells, compared to WNV-infected control cells. Furthermore, WNV-induced apoptosis was significantly heightened in Se-deficient cells and was contributed by loss of mitochondrial membrane potential and increased caspase activity. However, no significant difference was found in WNV copy numbers between control, Se-adequate and Se-deficient cell cultures. CONCLUSION: Overall results demonstrate that the in vitro Se-deficient model can be used to study responses of WNV to this essential nutrient. Although Se deficiency has no in vitro effect on WNV replication kinetics, adequate Se is presumably critical to protect WNV-infected cells against virus-induced cell death.

[In vitro inhibitory mechanism of respiratory syncytial virus with solution prescription of zhidanhuayu].

OBJECTIVE: To study the effective part of solution prescription of Zhidanhuayu (ZDHY) against respiratory syncytial virus (RSV) in vitro. METHODS: Observe the pathology of RSV to Hep-2 under the condition of different concentrations and each effective part of ZDHY. RESULTS: The concentration limit causing celluar toxicity of ZDHY is 5.5 mg/ml. The ZDHY failed to block the absorption of RSV to Hep-2 within this concentration, and consequently the cell fell into the full pathological changes. During the concentration of 2.75-5.50 mg/ml, the ZDHY directly destroyed virus array,meanwhile, the infected cells that treated by the medicine kept healthy also. CONCLUSION: ZDHY could not defend the infection of RSV, but is able to destroy the RSV directly and inhibit the RSV inhabiting in the cell.

[Microtubules are necessary for lamellae retraction of Vero cells].

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.

African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis.

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.

On the cyto-toxicity caused by quantum dots.

Quantum dots (QDs) such as CdSe QDs have been introduced as new fluorophores. The QDs conjugated with antibody are starting to be widely used for immunostaining. However there is still not sufficient analysis of the toxicity of QDs in the literature. Therefore we evaluated the cell damage caused by the quantum dots for biological applications. We performed cell viability assay to determine the difference in cell damage depending on the sizes and colors of mercapto-undecanoic acid (MUA) QDs and the cell types. The results showed that the cell viability decreased with increasing concentration of MUA-QDs. But in the case of Vero cell (African green monkey's kidney cell) with red fluorescence QD (QD640), the cell damage was less than for the others. Furthermore through the flow cytometry assay we found that this cell damage caused by MUA-QD turned out to be cell death after 4-6-hr incubation. From the two assays described above, we found that there is a range of concentration of MUA-QDs where the cell viability decreased without cell death occurring and thus we conclude that attention should be given when MUAQDs are applied to living organisms even in low concentrations.

Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine.

Entry of Japanese encephalitis virus (JEV) into cells was analysed by using the vertebrate cell line Vero. Vero cells were treated with chlorpromazine, nystatin or cytochalasin D, which inhibit clathrin- and caveola-dependent endocytosis, and macropinocytosis of the cells, respectively. Productive JEV infection was inhibited by pretreatment with chlorpromazine; the number of JEV antigen-positive cells was less than one-fifth of that in untreated cultures, but was not significantly decreased by pretreatment with nystatin or cytochalasin. Viral antigens were detected in the membrane fractions, but not in the endosome fractions from chlorpromazine-treated JEV-inoculated cells. When the cells were treated with chlorpromazine, clathrin heavy chain antigen and JEV antigen were not detected in cytoplasm by indirect immunofluorescence staining. These results indicate that JEV is taken up by cells through the clathrin-dependent endocytic pathway, and this process leads to infection.

Effect of temperature, medium composition, and cell passage on production of herpes-based viral vectors.

Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33 degrees C than at 37 degrees C and is further stabilized at 4 degrees C. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33 degrees C produced 2-fold higher amounts of vector than infected cells incubated at 37 degrees C, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33 degrees C than 37 degrees C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33 degrees C yielded similar levels regardless of passage number but was reduced at 37 degrees C as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.

Vero cells, but not oviductal cells, increase the hatching frequency and total cell count of mouse blastocysts partly by changing energy substrate concentrations in culture medium.

PURPOSE: To investigate the embryotrophic mechanisms of Vero and oviductal cells coculture. METHODS: Mouse embryos were cultured in Chatot, Ziomek, and Bavister medium (CZB), in modified CZB media (MM) with nutrient concentrations adjusted to that found in conditioned media after different periods of Vero cells or oviductal cells culture, in reconstituted medium (RM) containing the purified > 100-kDa components of Vero cell conditioned medium that had been reconstituted with CZB medium, and cocultured with Vero cells with an interposing membrane. RESULTS: The blastulation rate was not different among embryos cultured in different Vero-cell-derived MMs. Nine-hour Vero-cell-derived MM significantly increased the total cell number and hatching frequency of the embryos. There was no difference in these parameters with oviductal-cell-derived MMs. The RM of Vero cells did not possess embryotrophic activity. The presence of a porous membrane between Vero cells and embryos did not affect the embryotrophic activity of coculture. CONCLUSIONS: Vero cells, but not oviductal cells, improved mouse embryo development partly by modifying the energy substrate concentration in culture medium.

Bovine milk inhibits both adhesion of Helicobacter pylori to sulfatide and Helicobacter pylori-induced vacuolation of vero cells.

Adhesion of Helicobacter pylori to gastric mucosal cells is an initial important step in colonization and infection. To study adhesion, we investigated whether milk inhibits the adhesion of Helicobacter pylori to sulfatide, an acidic glycosphingolipid that exists in human gastric mucosa and to which Helicobacter pylori adheres. As a measure of functional significance, we also studied whether milk inhibits Helicobacter pylori-induced vacuolation of Vero cells. We used sulfatide-coated polystyrene plates and studied the effect of bovine milk on the adhesion of Helicobacter pylori to sulfatide. We used Vero cells for Helicobacter pylori-induced vacuolation. Bovine milk 100- to 200-fold diluted significantly inhibited both adhesion of Helicobacter pylori to sulfatide and Helicobacter pylori-induced vacuolation in Vero cells. Bovine milk significantly inhibited adhesion of Helicobacter pylori to MKN-45 cells and Lewis b antigen-coated polystyrene plates. In addition, these results suggest that bovine milk contains active substances that inhibit both adhesion of Helicobacter pylori to mucosa and vacuole formation. Bovine milk may have a protective effect on the gastric mucosa in Helicobacter pylori-associated gastritis.

Use of co-culture of human embryos on Vero cells to improve clinical implantation rate.

Co-culture of human embryos (n = 384 cycles) to the blastocyst stage using Vero cell monolayers was carried out between August 1995 and December 1997. A total of 2868 zygotes were co-cultured and 1027 embryos reached the blastocyst stage (blastocyst formation rate 35.8%). The blastocysts were frozen in 43.7% of patients. A mean of 1.8 blastocysts was transferred per patient and 95 pregnancies were obtained (pregnancy rate/cycle 24.7%). The blastocyst implantation rate was 23.6%. Miscarriage occurred in 15 patients (15.7%) and ectopic pregnancy in three (3.1%) patients. The multiple pregnancy rate was 32.6%. No differences were observed in the blastocyst rate between poor, normal or high response patients. Blastocyst formation was significantly lower when frozen donor spermatozoa were used. Significantly higher pregnancy rates per transfer and blastocyst implantation rates were attained when embryos were transferred on days 5 or 6 compared with day 7. No advantage was observed when co-culture was used in first cycle IVF patients, in comparison with conventional day 2 replacements. The use of blastocysts for preimplantation genetic diagnosis (PGD) increases the diagnostic reliability and widens diagnostic possibilities. A total of 215 cycles with frozen-thawed co-cultured blastocysts were carried out, with a pregnancy rate of 22.7% per replacement.

Co-cultured human embryos may be subjected to widely different microenvironments: pattern of growth factor/cytokine release by Vero cells during the co-culture interval.

This study was designed to identify and quantify concentrations of growth factors/cytokines released by Vero cells during the co-culture interval. The factors screened for in this preliminary investigation, namely platelet-derived growth factor (PDGF), transforming growth factor beta (TGFbeta), interleukin-6 (IL-6), leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) have each been identified to impact on early embryo development or are secreted by embryos themselves, suggesting an autocrine regulatory role. Vero cell culture supernatants were collected at 2, 3, 4, 5 and 6 days after seeding. Samples were assessed by enzyme-linked immunoassay for growth factor/cytokine secretion at each designated time interval. Conditioned medium from all days contained IL-6, PDGF and LIF. The concentration of IL-6 increased from 294 pg/well on day 2 to almost 1600 pg/well on day 6. PDGF also accumulated rapidly in co-culture wells, rising from 19-40 pg/well early in the culture period to around 500 pg/ well by day 6. In the second half of this study, medium supernatants from patients enrolled in our co-culture programme were analysed. Retrospective evaluation of medium supernatants collected at the time of transfer from co-cultures from 11 randomly selected patients showed considerable patient-to-patient variation in concentrations of secreted growth factors and cytokines. These findings indicate that during the co-culture interval embryos are exposed to a dynamic environment, with increasing concentrations of growth factors and cytokines. The positive effects of co-culture on embryo quality and in-vitro blastulation need to be balanced against the variation that this technique can potentially introduce into the embryo culture system.

Detection of apoptotic cells in Vero cell cultures inoculated with samples derived from fatal cases of Sarawak acute childhood viral infection.

Infectious agent(s) causing the fatal Sarawak acute childhood viral infection (SACVI) has not been identified. In the present study, results indicating that inocula prepared from the fatal cases of SACVI induced apoptosis in Vero cell cultures are presented. These findings suggest the possible involvement of apoptotic cellular responses in SACVI.

The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro.

Co-culture of human embryos with cell layers has generally shown that blastocyst formation rates are improved compared to routine culture in medium alone. In order to assess this further, we have additionally classified resulting blastocysts according to their morphology and secretion of human chorionic gonadotrophin (HCG). A total of 70 supernumerary human embryos from 15 patients were divided equally and randomly between two culture conditions: (i) co-culture with Vero cells; and (ii) culture in our routine medium. Embryo development and morphology were recorded for up to 14 days in culture. The results showed that embryos on Vero cells had a significantly higher blastocyst formation rate (P < 0.02) by or on day 6 of development than those in routine culture medium alone (77 and 46% respectively). For HCG analysis, the culture medium was changed in both culture systems on days 5, 7, 9, 12 and 14 of embryo development and analysed. Most embryos began to produce HCG between days 7 and 9, with HCG secretion being significantly higher from embryos on Vero cells between days 9 and 12 than from embryos in routine culture (P < 0.03). The morphology of the blastocysts obtained was related to their ability to hatch and produce HCG but was not significantly better for one type of culture system than for the other.