Growth In Vero Cells Research Articles

Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13.

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

Adaptation of a live-attenuated genotype I Japanese encephalitis virus to vero cells is associated with mutations in structural protein genes

The SD12-F120 is a live-attenuated genotype I strain of Japanese encephalitis virus (JEV) and was obtained by serial passage of wild-type strain SD12 on BHK-21 cells combined with multiple plaque purification and virulence selection in mice. The large scale production and vast clinical trials always demand ideal safety and efficacy profile of live-attenuated vaccines. In the present study, SD12-F120VC has undergone serial passaging of P1-P30 in WHO qualified Vero cells to assess the potential effect of adaptation to growth on Vero cells. The series of experiments showed that vaccine SD12-F120VC (Vero cell adapted) variants have consistently increased in peak virus titer compared to early passages and have good adaptation to growth in Vero cells. The animal experiments showed that Vero cell adapted SD12-F120VC variants have attenuation phenotype in suckling mice and the plaque morphology for all SD12-F120VC variants was small. Vaccination of mice with SD12-F120VC vaccine produced complete protection for homologous SD12 genotype I strain, but failed to give the complete protection of vaccinated mice against the challenge of heterologous N28 genotype III strain. In response to immunization of SD12-F120VC in mice, the neutralizing antibodies titer against homologous SD12-F120VC and SD12 (GI) was higher than heterologous N28 (GIII) strain. The prM protein has 6 amino acid substitutions, of which 5 amino acid changes were confined at the start of the pr domain in the ∼40 amino acids, and some mutations in the pr domain of prM might contribute to Vero cell adaptation. Our findings in this study are important for validation, evaluation and quality control study of live attenuated flaviviruses vaccines and show that Vero cells are a suitable substrate for the production of a safe and stable live-attenuated JEV vaccine.

Formulation and production of a blood-free and chemically defined virus production media for VERO cells.

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum‐containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum‐free media suitable for VERO‐based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum‐free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM‐10% FBS in this chemically defined medium while the plant hydrolysate‐containing medium demonstrated a slower doubling time in both two‐dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM‐10%FBS and better than the plant hydrolysate‐containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.

Phytochemical Characterization, In Vitro Antioxidant, Cytotoxic, and Antibacterial Effects of Aristolochia longa L.,

The aim of this study was devoted, in the one hand, to determine a preliminary phytochemical screening of methanolic and aqueous root extracts of Aristolochia longa L. In the other hand, polyphenols and flavonoids contents, in vitro antioxidant, antitumor, and antibacterial activities of the organic extract of A. longa were determined. Phytochemical screening was carried out using standard methods of precipitation and coloration reactions. The total phenol content and flavonoid contents were determined using the Folin-Ciocalteu method and Aluminum chloride colorimetric method, respectively. Antioxidant activity was studied using DPPH assay. The cytotoxic activity of extracts was evaluated against three cancerous cell lines using MTT assay, and antibacterial activity was tested against Escherichia coli, Pseudomonas aeruginosa, Rhodococcus equi, and Staphylococcus aureus using the agar well diffusion assay. Preliminary phytochemical screening for aqueous and ethanolic extracts revealed the presence of starch, tannins, flavonoids, coumarins, and anthocyanins. The total phenol content of A. longa extracts showed that the methanol extract has the highest polyphenol and flavonoid concentrations with 101.4 mg GAE (Gallic acid equivalent)/g and 54.21 mg QE (Quercetin equivalent)/g extract, respectively. The methanolic extract also exhibited the highest antioxidant capacity of DPPH (IC50 = 1.32 mg/mL). While hexane extract exhibited both a potent inhibitory effect on VERO cell growth with IC50 = 15.125 μg/mL and a total inhibitory effect on Staphylococcus aureus with inhibition zone 8.5 mm. The high content of polyphenols in A. longa explains their antioxidant, antitumor, and antibacterial activity. Thus, this plant could be a significant source of natural compounds in preventing the development of cancer.

Cytotoxicity of Alstonia scholaris (R.Br) bark on MCF-7 and Vero cell lines

Alstonia scholaris R.Br (pulai) is one of traditional forest plant to treat many kinds of disease such as arthritis, diabetes, hypertension, and hyperlipidaemia. On the other hand research about this species as anti cancer is very limited. This study aimed to determine in vitro cytotoxic activity of three fraction of A. Scholaris bark against breast cancer (MCF-7) and normal (vero) cells. Three fractions of A. Scholaris bark (n-hexane, chloroform, and ethanol fractions) derived from 70% ethanol crude extracted pulai bark. MTT (3- 4,5-dimethylthiazol-2- -2,5-diphenyl tetrazolium bromide) assay applied tot MCF-7 and vero cells. The result showed that n-hexane fraction had the strongest cytotoxic effect on MCF-7 cells, followed by chloroform and ethanol fraction with IC50 values 109.01; 163.33; and 264.19 µg/mL respectively, but ethanol fraction is the least toxic on vero cells growth compared to n-hexane and chloroform fraction with IC50 value 579.93; 459.47; and 396.24 µg/mL respectively. It’s suggested that ethanol fraction is the best recommended fraction as anticancer agent because it was the least toxicity to the growth of normal cells, but still toxic to cancer cell. It was concluded that ethanol fraction was the best fraction to breast cancer cells.

2'-Hydroxychalcones as an alternative treatment for trichomoniasis in association with metronidazole.

The treatment for trichomoniasis, based on 5'-nitroimidazol agents, has been presenting failures related to allergic reactions, side effects, and the emergence of resistant isolates. There are no alternative drugs approved for the treatment of these cases; thus, the search for new active molecules is necessary. In this scenario, chalcones have been extensively studied for their promising biological activities. Here, we presented the synthesis of three hydroxychalcones (3a, b, and c), in vitro and in silico analyses against Trichomonas vaginalis. The in vitro biological evaluation showed that hydroxychalcone 3c presented anti-T. vaginalis activity, with complete death in 12h of incubation at minimum inhibitory concentration (MIC) of 100μM. 3c showed a dose-dependent cytotoxicity against mammalian VERO cell line, but the association of 3c at 12.5μM and metronidazole (MTZ) at 40μM showed 95.31% activity against T. vaginalis trophozoites after 24h of exposure and did not affect the VERO cell growth, appearing to be a good alternative. In silico analysis by molecular docking showed that 3c could inhibit the activity of TvMGL (methionine gamma-lyase), TvLDH (lactate dehydrogenase), and TvPNP (purine nucleoside phosphorylase) affecting the T. vaginalis survival and also suggesting a different mechanism of action from MTZ. Therefore, these results propose that hydroxychalcones are promising anti-T. vaginalis agents and must be considered for further investigations regarding trichomoniasis treatment.

Preparation of cell culture scaffolds using polycaprolactone/quince seed mucilage

In the present study, the polymer obtained from Cydonia oblonga Miller seeds (quince seed mucilage (QSM)) in combination with polycaprolactone (PCL) was used for producing hybrid electrospun scaffolds as three-dimensional (3D) cell culture platforms. Various PCL/QSM ratios were tested to obtain a uniform product with an appropriate mean fiber diameter for cell growth. The chemical structures of the scaffolds were studied by FT-IR spectroscopy and their crystallinity was investigated by XRD. Fiber morphology studies by SEM revealed that the fiber mean diameters decrease upon increasing the QSM component of the electrospun compound. The PCL/QSM ratio of 60/40 exhibited desirable uniform bead-free nanofibers. The hydrophilicity of the scaffolds were explored by contact angle measurements, which indicated that more hydrophilic fibers are produced as the PCL/QSM ratio is decreased. Porosity and strength of the scaffolds were also studied, which showed the key role of PCL in mechanical strength of the nanofibers. The results of cell culture experiments using epithelial Vero cells on the scaffolds displayed their high capacity in cell growth and proliferation. It was revealed that the electrospun PCL/QSM based scaffolds with 3D structures and 75–150 nm mean fiber diameters are able to maximize adhesion and growth of Vero cells.

In vitro cytotoxicity of Alstonia scholaris (R.Br) bark on Vero and HeLa cell lines

Alstonia scholaris R.Br is one of forest plants that have medicinal properties used to treat various diseases. This study aimed to determine the activity of in vitro cytotoxicity of Alstonia scholaris bark against HeLa and Vero cells. Alstonia scholaris bark was macerated with 70% ethanol and fractionated using n-hexane and chloroform to obtain n-hexane, chloroform, and ethanol fraction. All three fractions were tested using MTT assay against HeLa (cervix cancer) and Vero (normal) cells. The results showed that chloroform fraction was the most toxic to HeLa cells with IC50 (125.06 μg/mL) and followed by ethanol fraction (200.07 μg/mL), and n-hexane fractions (238.47 μg mL) respectively. On the other hand, ethanol fraction was the least toxic to Vero cell growth with IC50 (579.93 μg/mL), followed by n-hexane fraction (459.47 μg/mL), and chloroform fraction was the most toxic (396.24 μg/mL). It was suggested that ethanol fraction was the best fraction because it had the least toxicity to a normal cell, but still had toxicity to HeLa cell.

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost.Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 106 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process.Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic.The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 106 cells/mL.

Kinetic model for adherent Vero cell growth and poliovirus production in batch bioreactors.

The production of poliovirus vaccines in adherent Vero cells in batch bioreactors usually consists of a two-step upstream process: (1) Vero cell cultivation on microcarriers and (2) poliovirus proliferation. In this study we developed a mathematical model to describe this two-step process. We introduced the calculation of the oxygen uptake rate (OUR) and a correction of measurement for the sampling effect in order to ensure the high quality data sets. Besides the data of the OUR, we selected glucose concentration, Vero cell concentration and the virus titer for daily in process control to evaluate the progress of the process. With the selected data sets, the described model can accurately describe poliovirus production by Vero cells. Several other regular in process control samples (e.g. lactate concentration, ammonia concentration, and amino acids concentration) were excluded from the model, simplifying the process control analysis and minimizing labor.

Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression.

The Orf virus (ORFV; Parapoxvirus) strain D1701 with an attenuated phenotype and excellent immunogenic capacity is successfully used for the generation of recombinant vaccines against different viral infections. Adaption for growth in Vero cells was accompanied by additional major genomic changes resulting in ORFV strain variant D1701-V. In this study, restriction enzyme mapping, blot hybridization and DNA sequencing of the deleted region s (A, AT and D) in comparison to the predecessor strain D1701-B revealed the loss of 7 open reading frames (ORF008, ORF101, ORF102, ORF114, ORF115, ORF116, ORF117). The suitability of deletion site D for expression of foreign genes is demonstrated using novel synthetic early promoter eP1 and eP2. Comparison of promoter strength showed that the original vegf-e promoter Pv as well as promoter eP2 display an up to 11-fold stronger expression than promoter eP1, irrespective of the insertion site. Successful integration and expression of the fluorescent marker genes is demonstrated by gene- and insertion-site specific PCR assays, fluorescence microscopy and flow cytometry. For the first time ORFV recombinants are generated simultaneously expressing transgenes in two different insertion loci. That allows production of polyvalent vaccines containing several antigens against one or different pathogens in a single vectored ORFV vaccine.

A DS-1 like G9P[6] human strain CDC-6 as a new rotavirus vaccine candidate

Human rotavirus vaccine Rotarix® (G1P[8]) has shown broad cross protection against homotypic and heterotypic Wa-like human rotavirus strains among children worldwide. This vaccine, however, appears to induce slightly less or non-consistent protection against DS-1 like rotavirus P[4] strains in some settings. In addition, children who are secretor or Lewis-negative and are vaccinated with Rotarix® often experience breakthrough infection with P[6] strains. By contrast, P[6] strains infect all children, irrespective of their secretor or Lewis status. In the present study, we report successful adaptation of a DS-1 like human rotavirus G9P[6] strain (CDC-6) to high growth in Vero cells and identify sequence changes that may be critical for enhanced growth in vitro and attenuation in vivo. This human G9P[6] strain could serve as a promising new and potential low-cost vaccine candidate for global use, particularly in targeted population with secretor or Lewis-negative status and high prevalent DS-1 like P[6] strains.

The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism.

The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2' cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.

Porcine epidemic diarrhea vaccine evaluation using a newly isolated strain from Korea

Porcine epidemic diarrhea virus (PEDV) infects pigs and causes an enteric disease that is characterized by vomiting and watery diarrhea. PEDV outbreaks have a tremendous financial impact on the worldwide pork industry. In South Korea, the incidence of PEDV has continued despite nationwide use of attenuated and inactivated vaccines, raising questions regarding the current vaccines’ efficacy and the need for new vaccine development. In the present study, we isolated a new Korean PEDV epidemic strain, PED-CUP-B2014, in Vero cells. Phylogenetic analysis of the spike gene demonstrated that the PED-CUP-B2014 belongs in genogroup G2b and is close to PEDVs currently circulating in many countries including the United States, and is distinct from many current vaccine strains. Upon serial passages into Vero cells, PED-CUP-B2014 adapted to Vero cells, which was evidenced as higher virus growth in Vero cells and confirmed lower virulence in suckling piglets. The administration of the inactivated 65-passaged PED-CUP-B2014 to sows greatly increased the survival rate of their offspring and significantly reduced diarrhea severity after PEDV challenge. Higher serum/colostrum PEDV-specific antibodies and higher neutralizing titers were shown in sows vaccinated with PED-CUP-B2014 compared to unvaccinated sows or sows administered commercial PEDV vaccine. Altogether, our data demonstrated that the newly isolated PEDV strain conferred critical passive immune protection to pigs against epidemic PEDV infection.

A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells.

Vaccination is the primary strategy for influenza prevention and control. However, egg-based vaccines, the predominant production platform, have several disadvantages, including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted the development of cell-based vaccines, which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR-based mutagenesis strategy, we identified a Y161F mutation in hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold but also increased its thermostability without changing its original antigenic properties. The vaccine produced from the Y161F mutant fully protected mice against lethal challenge with wild-type A(H1N1)pdm09. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, the introduction of the Y161F mutation into HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses.IMPORTANCE Although a promising complement to current egg-based influenza vaccines, cell-based vaccines have one large challenge: high-yield vaccine seeds for production. In this study, we identified a molecular signature, Y161F, in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two cell lines commonly used for influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability but also enhanced its binding affinity for α2,6- and α2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.

Ribavirin and boceprevir are able to reduce Canine distemper virus growth in vitro

Canine distemper virus (CDV) is a major infectious disease of dogs. Although vaccines were successful to control CDV spread in canine population, the disease is still common and may pose a threat to unvaccinated dogs. In the attempt to develop specific anti-viral therapeutic tools, the efficacy of several molecules against CDV has been investigated in vitro. In this study the antiviral efficacy in vitro against CDV of ribavirin and boceprevir alone or in combination was evaluated.CDV growth in VERO cells was inhibited by ribavirin, by boceprevir and by a combination of the two molecules at non-cytotoxic concentrations, as evaluated by end-point viral titration in cell monolayers and by quantification of viral RNA using quantitative RT-PCR. By end-point titration, a statistically significant reduction in CDV replication was observed only using ribavirin and boceprevir in combination. By quantitative RT-PCR, a significant reduction of viral growth was observed either in cells treated with ribavirin or boceprevir or with both the two molecules. The association of ribavirin or boceprevir was able to decrease CDV growth by up to 3.4458 logs with respect to untreated infected cells, chiefly at the highest virus dilutions. The results obtained in this study may constitute an important basis for the development of CDV therapies.

Carbohydrates–chitosan composite carrier for Vero cell culture

In this study, carbohydrate-chitosan composite including glucose-chitosan, sucrose-chitosan and starch-chitosan with varied carbohydrate concentrations were prepared as carriers for Vero cell culture. Our results show that among these composites, 30% starch-chitosan composite (STC) were the best carriers for the growth of Vero cells. The initial number of attached cells on the surface of composite carriers did not have any significant effect on subsequent cell production. A higher glucose level in the growth medium during the exponential phase of cell growth, however, played an important factor for cell production. Vero cells on the STC carriers were able to convert starch inside the composite carriers into glucose and further utilized the glucose for their growth. Moreover, by crosslink with serum the STC carriers supported an even better cell production in the normal medium without adding fetal bovine serum, as well as a good extracellular virus production. The STC composite is therefore a promising alternative carrier for Vero cell culture.

Ultraviolet/ozone treated polystyrene microcarriers for animal cell culture

BACKGROUND Microcarrier based cell culture offers many advantages when compared with conventional cell cultures, and this technology has been widely used in the production of many important biological materials. In this work, microcarriers were produced using materials and methods that are relatively simple and low-cost. RESULTS Polystyrene (PS) microspheres were prepared by using an emulsion solvent–evaporation method and the effects of process conditions on the size of microspheres were investigated. The mean size of microspheres decreased with increasing agitation speed, concentration of polyvinyl alcohol (PVA) and polymer to solvent ratios but increased with increasing oil to water phase ratios. To enhance its cell attachment abilities, surfaces of PS microspheres were loaded with oxygen functional groups by using an ultraviolet/ozone (UVO3) system. Analysis by Fourier transform infrared (FTIR), energy dispersive X-ray spectroscopy (EDX), toluidine blue O (TBO) assay and relative contact angle measurements showed increased surface oxygen concentration after UVO3 treatment. Results from spinner flask culture revealed that UVO3 treated PS microspheres support the growth of Vero cells to high cell density. CONCLUSION UVO3 treatment successfully rendered the hydrophobic surface of PS microspheres hydrophilic. This newly developed microcarrier could serve as a low-cost alternative to commercial microcarriers available in the market today. © 2015 Society of Chemical Industry

Evaluation of physicochemical and biological properties of chitosan/poly (vinyl alcohol) polymer blend membranes and their correlation for Vero cell growth

The blend membranes with varying weight ratios of chitosan/poly (vinyl alcohol) (CS/PVA) (1:0, 1:1, 1:2.5, 1.5:1, 1.5: 2.5) were prepared using solvent casting method and were evaluated for their potential application in single-use membrane bioreactors (MBRs). The physicochemical properties of the prepared membranes were investigated for chemical interactions (FTIR), surface morphology (SEM), water uptake, protein sorption (qe), ammonia sorption and growth kinetics of Vero cells. CS/PVA blend membrane having weight ratio of 1.5:1 had shown enhanced membrane flexibility, reduced water uptake, less protein sorption and no ammonium sorption compared to CS membrane. This blend membrane also showed comparatively enhanced higher specific growth rate (0.82/day) of Vero cells. Improved physicochemical properties and growth kinetics obtrude CS/PVA (1.5:1) as a potential surface for adhesion and proliferation with possible application in single use membrane bioreactors. Additionally, new insight explaining correlation between water holding (%) of CS/PVA (1.5:1) blend membrane and doubling time (td) of Vero cells is proposed.

Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway.

Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with ¼ IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer.