Suspension-cultured Cells Research Articles

Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum.

The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.

A chitin deacetylase from the endophytic fungus Pestalotiopsis sp. efficiently inactivates the elicitor activity of chitin oligomers in rice cells.

To successfully survive in plants, endophytes need strategies to avoid being detected by the plant immune system, as the cell walls of endophytes contain easily detectible chitin. It is possible that endophytes “hide” this chitin from the plant immune system by modifying it, or oligomers derived from it, using chitin deacetylases (CDA). To explore this hypothesis, we identified and expressed a CDA from Pestalotiopsis sp. (PesCDA), an endophytic fungus, in E. coli and characterized this enzyme and its chitosan oligomer products. We found that when PesCDA modifies chitin oligomers, the products are partially deacetylated chitosan oligomers with a specific acetylation pattern: GlcNAc-GlcNAc-(GlcN)n-GlcNAc (n ≥ 1). Then, in a bioactivity assay where suspension-cultured rice cells were incubated with the PesCDA products (processed chitin hexamers), we found that, unlike the substrate hexamers, chitosan oligomer products no longer elicited the plant immune system. Thus, this endophytic enzyme can prevent the endophyte from being recognized by the plant immune system; this might represent a more general hypothesis for how certain fungi are able to live in or on their hosts.

Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in exvivo systems than in invitro cell cultures. I have therefore established an exvivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p=0.0083) and P2 (p<0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0±0.66% for suspension cells, 46.4±2.9% for monolayer cells, and 127.64±0.90% for pellets (p<0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over invitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such exvivo explants might serve as a better model system to mimic clinical situations.

Regulation of desiccation tolerance and long-term preservation of suspension-cultured cells of citrus

Regulation of desiccation tolerance and long-term preservation of suspension-cultured cells of citrus

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures

ObjectiveTo develop a microwell suspension platform for the adaption of attached stem cell differentiation protocols into mixed suspension culture.ResultsWe adapted an adherent protocol for the retinal differentiation of human induced pluripotent stem cells (hiPSCs) using a two-step protocol. Establishing the optimum embryoid body (EB) starting size and shaking speed resulted in the translation of the original adherent process into suspension culture. Embryoid bodies expanded in size as the culture progressed resulting in the expression of characteristic markers of early (Rx, Six and Otx2) and late (Crx, Nrl and Rhodopsin) retinal differentiation. The new process also eliminated the use of matrigel, an animal-derived extracellular matrix coating.ConclusionsShaking microwells offer a fast and cost-effective method for proof-of-concept studies to establish whether pluripotent stem cell differentiation processes can be translated into mixed suspension culture.

Skin-derived stem cells as a source of primordial germ cell- and oocyte-like cells.

The skin is a unique organ that contains a variety of stem cells for the maintenance of skin homeostasis and the repair of skin tissues following injury and disease. Skin-derived stem cells (SDSCs) constitute a heterogeneous population of stem cells generated in vitro from dermis, which can be cultured as spherical aggregates of cells in suspension culture. Under certain in vitro or in vivo conditions, SDSCs show multipotency and can generate a variety of neural, mesodermal, and endodermal cell types such as neurons, glia, fibroblasts, adipocytes, muscle cells, chondroblasts, osteoblats, and islet β-cell-like cells. SDSCs are likely derived from multipotent stem cells located in the hair follicles that are, in turn, derived from embryonic migratory neural crest or mesoderm cells. During the past decade, a wave of reports have shown that germ cells can be generated from various types of stem cells. It has been shown that SDSCs are able to produce primordial germ cell-like cells in vitro, and even oocyte-like cells (OLCs). Whether these germ cell-like cells (GCLCs) can give rise to viable progeny remains, however, unknown. In this review, we will discuss the origin and characteristics of SDSCs from which the GCLC are derived, the possible mechanisms of this differentiation process, and finally the prospective biomedical applications of the SDSC-derived GCLCs.

Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87% of the basic fibroblast growth factor and 80% of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1±0.1)×106cellsmL-1 was obtained, with 99.5±0.2% OCT 3/4 and 78.3±1.1% TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

O-glycans and O-glycosylation sites of recombinant human GM-CSF derived from suspension-cultured rice cells, and their structural role

Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O-glycans comprising arabinose and galactose. Eight O-glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O-glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O-glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O-glycosylation of rhGM-CSF derived from plant cells.

Variation in the flavonolignan composition of fruits from different Silybum marianum chemotypes and suspension cultures derived therefrom

Mature fruits collected from different milk thistle (Silybum marianum (L.) Gaertn.) plants, grown in various habitats in Europe, were analysed for silymarin content and variation in component composition. Two different German and Polish cultivars each, as well as fruits from Hungary and Bulgaria have been compared with respect to their ratio of flavonolignan regioisomers. Besides differences in total silymarin content (0.8%–4.9%), three distinct chemotypical variations in fruit flavonolignan regioisomer composition in the cultivars have been observed. Although the differences in the diastereomer ratios of silybin A/B and isosilybin A/B were not significant, they never appeared in a 1:1 ratio. In vitro cultures have been established from seedlings of three typical chemotypes for further insights into flavonolignan content and composition in suspension cultures and the release of these specialized compounds to the extracellular space. The differences in the three Silybum marianum chemotypes were also observed in the composition of the intracellular silymarin of suspension-cultured cells. Silymarin components released to the cell culture medium, however, showed a highly differing composition with only low amounts of silychristin and silydianin. Assays with crude protein extracts prepared from suspension cells or habituated medium of these three chemotypes did not result in differences in silymarin content or composition. In in vitro assays the formation of the regioisomers silydianin and silychristin were strongly influenced by the taxifolin:coniferyl alcohol concentration ratio.

In vitro production of baculoviruses: identifying host and virus genes associated with high productivity.

Baculoviruses are recognized as viral workhorses of biotechnology, being used for production of vaccines, complex recombinant proteins, gene delivery vectors' and safe biological pesticides. Improving production yields and understanding the interactions of the virus and its host cell are important aspects of ensuring baculovirus-based processes are commercially competitive. This study aims at potential optimization of host cells used in in vitro virus production by systemically investigating changes in host gene expression in response to virus replication and transcription inside host cells. The study focuses on in vitro interactions of the Helicoverpa armigera virus with Helicoverpa zea insect cells. We used 22 genome-wide microarrays to simultaneously measure both virus and host genes in infected cells in multiple batch suspension cultures, representing high- and low-producing infection conditions. Among 661 differentially expressed genes, we identified a core set of 59 host genes consistently overexpressed post infection, with strong overrepresentation of genes involved in retrotransposition, protein processing in the endoplasmic reticulum, and ubiquitin-mediated proteolysis. Applying a whole genome correlation network analysis to link gene expression to productivity, we revealed 18 key genes significantly associated to virus yield. In addition, this study is among the first to perform a genome-wide expression study for a major baculovirus group II strain, the H. armigera virus, extending current understanding of baculovirus-insect interactions, which mainly focuses on group I viruses.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements.

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell's metabolic activity for the biosynthesis of secondary wall polymers.

Time course of programmed cell death, which included autophagic features, in hybrid tobacco cells expressing hybrid lethality

PCD with features of vacuolar cell death including autophagy-related features were detected in hybrid tobacco cells, and detailed time course of features of vacuolar cell death were established. A type of interspecific Nicotiana hybrid, Nicotiana suaveolens×N. tabacum exhibits temperature-sensitive lethality. This lethality results from programmed cell death (PCD) in hybrid seedlings, but this PCD occurs only in seedlings and suspension-cultured cells grown at 28°C, not those grown at 36°C. Plant PCD can be classified as vacuolar cell death or necrotic cell death. Induction of autophagy, vacuolar membrane collapse and actin disorganization are each known features of vacuolar cell death, but observed cases of PCD showing all these features simultaneously are rare. In this study, these features of vacuolar cell death were evident in hybrid tobacco cells expressing hybrid lethality. Ion leakage, plasma membrane disruption, increased activity of vacuolar processing enzyme, vacuolar membrane collapse, and formation of punctate F-actin foci were each evident in these cells. Transmission electron microscopy revealed that macroautophagic structures formed and tonoplasts ruptured in these cells. The number of cells that contained monodansylcadaverine (MDC)-stained structures and the abundance of nine autophagy-related gene transcripts increased just before cell death at 28°C; these features were not evident at 36°C. We assessed whether an autophagic inhibitor, wortmannin (WM), influenced lethality in hybrid cells. After the hybrid cell began to die, WM suppressed increases in ion leakage and cell deaths, and it decreased the number of cells containing MDC-stained structures. These results showed that several features indicative of autophagy and vacuolar cell death were evident in the hybrid tobacco cells subject to lethality. In addition, we documented a detailed time course of these vacuolar cell death features.

Molecular characterization of an elicitor-responsive 3-hydroxy-3-methylglutaryl coenzyme A reductase gene involved in oleanolic acid production in cell cultures of Achyranthes bidentata

Achyranthes bidentata contains a rich source of important pharmaceutically active triterpene acids including oleanolic acid (OA) as a major one. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme to provide mevalonate for biosynthesis of triterpene acids. In this study, in order to develop a sustainable source of OA, cell suspension cultures were established from shoot cultures of A. bidentata, and a full length cDNA encoding HMGR (designated as AbHMGR) was cloned and characterized. The cDNA contained 2078 nucleotides with a complete open reading frame of 1593 nucleotides, which was predicted to encode a peptide of 530 amino acids. Expression analysis by real-time PCR revealed that AbHMGR mRNA was abundant in A. bidentata roots, stems and leaves. When cultivated in Murashige and Skoog medium supplemented with 1.5 mg/L 1-naphthlcetic acid (NAA) and 1.5 mg/L 6-benzyladenine (6-BA), cells in suspension culture grew rapidly, yielding OA (100.9 mg/L) after 15 days. OA content in cell cultures was increased under the elicitation of methyl jasmonate (MeJA), yeast elicitor (YE) or cadmium chloride (CdCl2). The ultrahigh production of OA was achieved to 371.8 mg/L, a 5.4-fold of the control after 2-day treatment of 0.2 mM MeJA in the cell cultures. Quantitative real-time PCR analysis showed that AbHMGR was expressed at a higher level under the elicitation of MeJA or YE. Our results suggested that OA production may be the result of the up-regulated expression of AbHMGR under the treatment of various elicitors.

Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota

In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL−1 and 477.46μgL−1, respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL−1), lutein (25949.54μgL−1) and α-tocopherol (8063.82μgL−1) chlorophyll a (1625.13μgL−1) and b (9.958 (9958.33μgL−1) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway.Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture.

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2- to 10-fold increase) without any influence on pluripotency. In addition, albumin-associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009-1016, 2016.

Multiple Patterns of Regulation and Overexpression of a Ribonuclease-Like Pathogenesis-Related Protein Gene, OsPR10a, Conferring Disease Resistance in Rice and Arabidopsis.

An abundant 17 kDa RNase, encoded by OsPR10a (also known as PBZ1), was purified from Pi-starved rice suspension-cultured cells. Biochemical analysis showed that the range of optimal temperature for its RNase activity was 40–70°C and the optimum pH was 5.0. Disulfide bond formation and divalent metal ion Mg2+ were required for the RNase activity. The expression of OsPR10a::GUS in transgenic rice was induced upon phosphate (Pi) starvation, wounding, infection by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), leaf senescence, anther, style, the style-ovary junction, germinating embryo and shoot. We also provide first evidence in whole-plant system, demonstrated that OsPR10a-overexpressing in rice and Arabidopsis conferred significant level of enhanced resistance to infection by the pathogen Xoo and Xanthomona campestris pv. campestris (Xcc), respectively. Transgenic rice and Arabidopsis overexpressing OsPR10a significantly increased the length of primary root under phosphate deficiency (-Pi) condition. These results showed that OsPR10a might play multiple roles in phosphate recycling in phosphate-starved cells and senescing leaves, and could improve resistance to pathogen infection and/or against chewing insect pests. It is possible that Pi acquisition or homeostasis is associated with plant disease resistance. Our findings suggest that gene regulation of OsPR10a could act as a good model system to unravel the mechanisms behind the correlation between Pi starvation and plant-pathogen interactions, and also provides a potential application in crops disease resistance.

A rapid and efficient method to study the function of crop plant transporters in Arabidopsis.

Iron (Fe) is an essential micronutrient for humans. Fe deficiency disease is widespread and has led to extensive studies on the mechanisms of Fe uptake and storage, especially in staple food crops such as rice. However, studies of functionally related genes in rice and other crops are often time and space demanding. Here, we demonstrate that transgenic Arabidopsis suspension culture cells and Arabidopsis plants can be used as an efficient expression system for gain-of-function study of selected transporters, using Fe transporters as a proof-of-principle. The vacuolar membrane transporters OsVIT1 and OsVIT2 have been described to be important for iron sequestration, and disruption of these two genes leads to Fe accumulation in rice seeds. In this study, we have taken advantage of the fluorescent-tagged protein GFP-OsVIT1, which functionally complements the Fe hypersensitivity of ccc1 yeast mutant, to generate transgenic Arabidopsis suspension cell lines and plants. GFP-OsVIT1 was shown to localize on the vacuolar membrane using confocal microscopy and immunogold EM. More importantly, the Fe concentration, as well as the concentration of Zn, in the transgenic cell lines and plants were significantly increased compared to that in the WT. Taken together, our study shows that the heterologous expression of rice vacuolar membrane transporter OsVIT1 in Arabidopsis system is functional and effectively enhances iron accumulation, indicating an useful approach for studying other putative transporters of crop plants in this system.

Analysis of the Proteins Secreted from the Oryza meyeriana Suspension-Cultured Cells Induced by Xanthomonas oryzae pv. oryzae.

Oryza meyeriana, a wild species of rice from China, shows high resistance to Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial blight, one of the most serious rice pathogens. To better understand the resistance mechanism, a proteomic study was conducted to identify changes in the proteins secreted in embryo cell suspension cultures in response to Xoo. After two-dimensional difference gel electrophoresis (2D-DIGE), 72 differentially expressed protein spots corresponding to 34 proteins were identified by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry. Of the 34 proteins, 10 were up regulated and 24 down regulated. The secreted proteins identified were predicted to be involved in various biological processes, including signal transduction, defense, ROS and cell wall modification. 77% of the 34 proteins were predicted to have a signal peptide by Signal P. Quantitative Real-Time PCR showed that transcript levels of 14 secreted proteins were not well correlated with secreted protein levels. Peroxidase activity was up regulated in both O. meyriana and susceptible rice but was about three times higher in O. meyeriana. This suggests that peroxidases may play an important role in the early response to Xoo in O. meyeriana. These results not only provide a better understanding of the resistance mechanism of O. meyeriana, but have implications for studies of the interactions between other plants and their pathogens.

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150–200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.

Taxanes content and cytotoxicity of hazel cells extract after elicitation with silver nanoparticles

The toxicity of silver nanoparticles (AgNPs) has been attributed to the generation of Ag+ ions as well as production of ROS. The latter can elicit defensive response of plant cells in different ways e.g., enhancement of secondary metabolite productions. In the present study this hypothesis was evaluated by assessment of taxanes production by suspension-cultured hazel (Corylus avellana L.) cells after treatment with AgNPs. The cells were treated with different concentrations of AgNPs (0, 2.5, 5, and 10 ppm), in their logarithmic growth phase (d7) and were harvested after 1 weak. The growth of cells and their membrane integrity decreased but extracellular electro conductivity and total dissolved solids increase by AgNPs (probably due to loosening of cell membrane). Treatment of hazel cells with AgNPs (in particular of 5 ppm) rapidly and remarkably increased the yields of two major taxanes, i.e., Taxol and baccatin III; so that 24 h of the treatment their contents reached to 378% and 163% of the control, respectively. Increase of Taxanes was accompanied by the increase of total soluble phenols. The extracts of AgNPs-treated cells were able to inhibit the growth of cancerous HeLa cells and reduce their viability to 60% of the control. The results suggest the elicitation of suspension-cultured hazel cells with AgNPs as a procedure for rapid enhancement of anticancer taxanes biosynthesis by the cells.