Growth Of Cell Suspensions Research Articles

Endophytic fungus-suspension cell co-culture: A new strategy for more rapid and sustainable screening of plant beneficial microorganisms

Microbial fertilizers are increasingly used as alternatives to conventional fertilizers, while the screening process for beneficial microorganisms is inefficient and time-consuming, often taking months or years. This study aimed to evaluate whether the endophytic fungus-suspension cell co-culture strategy could be used for rapid screening of plant beneficial microorganisms. Therefore, we investigated the effects and molecular mechanisms of Fusarium oxysporum GU-7 and GU-60 on the growth and accumulation of secondary metabolites in Glycyrrhiza uralensis suspension cells and potted seedlings. GU-7 significantly promoted the growth of suspension cells and potted seedlings, which may be related to its strong phosphorus-solubilizing and indole-3-acetic acid production capacity. In contrast to GU-60, GU-7 promoted the accumulation of flavonoids and glycyrrhizin at different co-culture times and growth times. Both strains accelerated the accumulation of liquiritigenin, liquiritin, and glycyrrhizin in suspension cells after 24 h of co-culture. Meanwhile, they differentially altered the expression of critical genes. The defense response of suspension cells was enhanced at 72 h, the antioxidant enzyme activity and flavonoid aglycone content were increased and the flavonoid glycoside content was decreased. RNA-seq revealed that the two strains had five times as many glycoside hydrolases as glycosyltransferases and differed in biological processes and biosynthetic pathways involved. Conclusively, the effects of these two strains on suspension cells were consistent with those on potted seedlings, while different strains produced different effects. These results suggest that co-culture with suspension cells is a promising strategy for rapid screening of beneficial microorganisms, even for conspecific fungi. GU-7 has the potential to be further developed as a microbial fertilizer to improve the growth and secondary metabolite accumulation of cultivated licorice.

A disposable impedance-based sensor for in-line cell growth monitoring in CAR-T cell manufacturing

This paper presents the development of low-cost, disposable impedance-based sensors for real-time, in-line monitoring of suspension cell culture. The sensors consist of electrical discharge machining (EDM) cut aluminum electrodes and polydimethylsiloxane (PDMS) spacers, both of which are low-cost materials that can be safely disposed of. Our research demonstrates the capability of these low-cost sensors for in-line, non-invasive monitoring of suspension cell growth in cell manufacturing. We use a hybrid equivalent circuit model to extract key features/parameters from intertwined impedance signals, which are then fed to a novel physics-inspired (gray-box) model designed for α-relaxation. This model determines viable cell count (VCC), a critical quality attribute (CQA) in cell manufacturing. Predicted VCC trends are then compared with image-based cell count data to verify their accuracy.

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth.Key points• Less and more efficient glucose utilization for suspension cell growth• Concerted alteration of metabolic enzyme activity and protein expression• Protein candidates to interfere glycolytic activity in MDCK cells

Application of nanoparticles (ZnO, TiO2 and CuO), a new opportunity for the stimulation of cell growth and azadirachtin production in cell suspension culture of Azadirachta indica

Nanoparticles have unique physicochemical properties and provide great opportunities in plant science studies. In this study, we investigated the impact of ZnO, TiO2 and CuO nanoparticles (0, 20, 40, 60, and 80 mg/L) and sampling times (2, 4, and 6 days) on cell suspension growth and azadirachtin accumulation and production. Factorial experiments based on a completely randomized design with three replications were used. Results demonstrated that different nanoparticles had a different effect on the studied characters. When ZnO nanoparticles were used, the highest fresh (540.73 g/L), dry cell weight (15.93 g/L), azadirachtin accumulation (5.15 mg/g DW) and production (68.27 mg/L) were obtained at control condition, 80 and 40 mg/L ZnO nanoparticles, and control condition after 6 days, respectively. The highest amount of fresh (526.95 g/L) and dry (17.05 g/L) cell weight and azadirachtin production (82.21 mg/L) and accumulation (5.93 mg/g DW) were observed in 20 mg/L TiO2 nanoparticles, 40 mg/L of TiO2 nanoparticles after 2 days, 20 mg/L TiO2 nanoparticles in 4 days and 60 mg/L of TiO2 nanoparticles, respectively. With applying CuO nanoparticles, the highest fresh cell weight and azadirachtin accumulation were 422.59 g/L and 4.00 mg/L, achieved in control conditions respectively. Also, the highest amount of azadirachtin production was 68.27 mg/L, observed in control conditions on the 6th day of treatment. It seems that suitable cell growth, except in some cases, occurred in the absence of elicitors, but azadirachtin accumulation and production were stimulated by nanoparticles treatment. However, the results showed that the CuO nanoparticles caused a decrease in overall azadirachtin accumulation and production in the cells.

Spectral quality as an elicitor of bioactive compound production in Solanum aculeatissimum JACQ cell suspension

Solanum aculeatissimum Jacq. is a common plant in much of Brazil. Despite containing metabolites with a wide range of pharmacological applications, there are few tissue culture reports for this plant. The possibility of large-scale in vitro production of this material has significant biotechnological potential. Therefore, the objective of this study was to investigate the effect of light conditions on the growth of cells in suspension, observing the production and yield of biomass and bioactive compounds and the enzymatic behavior. Calli obtained from leaf segments were cultured in solid medium supplemented with 1 mg L−1 of 2,4-D, 2.5 mg L−1 kinetin, pH 5.7, in the dark. After 110 days of subculture, the calli were transferred to liquid medium. Cells were kept in the dark under agitation at 110 rpm and 25 °C and subcultured every 30 days. After 90 days of culture, 20 mL aliquots of cell suspension were added to flasks containing approximately 20 mL of medium (1:1) and cultured at different wavelengths (white, green, blue, red, and blue/red) under a photoperiod of 16 h with irradiance of 50 μmol m−2 s−1) and in the absence of light. The experiment was performed in a 6 × 6 factorial design (light condition × culture time). The cell cultures showed viability throughout the entire cycle, and chlorogenic and ferulic acids, orientin, quercitrin and, in higher amounts, quercetin, were detected in the first 7 days of culture. There was an increase in superoxide dismutase and catalase and a decrease in ascorbate peroxidase after exposure to different light conditions; for phenylalanine ammonia lyase, no differences were observed. The different light conditions were not sufficient to trigger responses in the concentrations of bioactive compounds, despite the detection of increased levels of the enzymes involved in cellular homeostasis.

Phytochemical Screening of Callus and Cell Suspensions Cultures of Thevetia peruviana

Abstract Thevetia peruviana is an ornamental shrub grown-up in many tropical region of the world. This plant produces secondary metabolites with biological properties of interest for the pharmaceutical industry. The objective was to determine the secondary metabolites profile of callus and cell suspension cultures of T. peruviana and compare them with those from explant (fruit pulp). Extracts in 50% aqueous ethanol and ethyl acetate were prepared. The phytochemical analysis was performed using standard chemical tests and thin layer chromatography. In addition, total phenolic and flavonoids compounds (TPC and TFC), total cardiac glycosides (TCG) and total antioxidant activity (TAA) was determined during the cell suspension growth. Phenolic chemical profile was also analyzed by high performance liquid chromatography (HPLC). Common metabolites (alkaloids, amino acids, antioxidants, cardiac glycosides, leucoanthocyanidins, flavonoids, phenols, sugars and triterpenes) were detected in all samples. The maximum production of extracellular TCG, TPC, TFC and TAA in cells suspensions were at 6-12 days; in contrast, intracellular content was relatively constant during the exponential grown phase (0 to 12-days). HPLC analysis detected one compound with retention time at 11.6 min; this compound was tentatively identified as dihydroquercetin, a flavonoid with anti-cancer properties. These results provide evidence on the utility of the in vitro cell cultures of T. peruviana for valuable pharmaceutical compounds production.

New biological trends on cell and callus growth and azadirachtin production in Azadirachta indica.

Azadirachtin is an important secondary metabolite from Azadirachta indica used as a natural biopesticide. This study is the first comprehensive report concerning the influence of plant growth regulators on callus induction, cell suspension growth, and azadirachtin accumulation and production in cell suspension cultures of A. indica. We investigated the effect of plant growth regulators including different types of auxins and cytokinins and their combinations on callus induction, cell suspension growth, and azadirachtin accumulation and production. The highest percentage of callusing (100%) obtained at different combinations of plant growth regulators on MS medium supplemented with 1mg/L picloram and 2mg/L kinetin and the highest fresh weight of callus (264.50mg) was observed in MS medium containing 1.5mg/L NAA and 3mg/L kinetin. In cell suspension cultures, the maximum cell density, SCV, and PCV were 2.44 × 106cells per mL, 97.95%, and 81.46%, respectively, obtained in the MS medium containing 1.5mg/L 2,4-D and 3mg/L zeatin riboside. The highest average growth rate (0.25days) was on MS medium containing 1.5mg/L NAA and 3mg/L zeatin riboside. The MS medium supplemented with 1mg/L picloram and 2mg/L kinetin produced the highest amount of fresh cell weight (493.02g/L), dry cell weight (77.27g/L), azadirachtin accumulation (3.69mg/gDW), and azadirachtin production (285.64mg/L). The results showed that all measured indices had positive correlation with together except FCW and DCW with azadirachtin accumulation.

Dissecting lipid accumulation of microalgae Nannochloropsis oculata using fluorescent image analysis

Cell suspension of Nannochloropsis oculata was cultured in a modified f/2 medium to study the changes of lipid content during phases of growth. The growth of cell suspension was determined by measuring the cell density and diameter under light microscope. To observe and evaluate the accumulation of lipid droplets in microalgae cells, lipid droplets were stained with Nile Red fluorescent dye then examined under fluorescence microscope and the obtained images were analyzed using Fiji ImageJ, an image processing program. The cell density increased quickly at the first 6 days of culture while cell diameter reached the highest value at the 8th day and 20th day of culture. The presence of lipid droplets in the cells could be observed from the 20th day of culture. The size of lipid droplets was gradually increased after 60 days. Treatment of depleted nitrogen for 4 days resulted an increase in the accumulation of lipid. The intracellular lipid accumulation during phases of growth of the cell suspension under nitrogen-depleted conditions was also discussed.

Optimization of active compounds production by interaction between nitrate and copper in Sphaeralcea angustifolia cell suspension using Response Surface Methodology

Cells in suspension from Sphaeralcea angustifolia produce compounds with anti-arthritic activity (scopoletin, tomentin, and sphaeralcic acid) in Murashige and Skoog (MS) medium with 2.74 mM of total nitrate content. The effect of nitrate and copper contents in the MS medium on the growth of cell suspension and the production of active compounds were tested by means of 2k factorial design (FD) and Central Composite Design (CCD). The growth rate and the duplication time were statically similar and the maximal biomass (9–11 days) was improved by increases of the nitrate concentration. Highest contents of coumarins (4137.00 µg L−1) and sphaeralcic acid (1441.00 µg L−1) were obtained in the culture media by the interaction of 2.74 mM of nitrates and 2 µM of copper at 2 and 4 days; sphaeralcic acid contents were similar to those detected intracellularly after 9 and 11 days of culture. According to the CCD, highest contents of coumarins (4008.00 µg L−1) and sphaeralcic-acid (6107.00 µg L−1) could be obtained with 2.35 µM of copper with 2.42 mM of total nitrate, this condition did not affect the cell growth. The coumarin content is 40-fold and that of the sphaeralcic-acid is 30-fold higher than those detected in biomass. The S. angustifolia cell suspension could be cultured in a stirred tank bioreactor in a batch or in a continuous manner for the production of scopoletin, tomentin, and sphaeralcic-acid.

Changes in Cytomorphology, Expression of Retinoblastoma Related Gene, and Superoxide Dismutase Enzyme Activity in Maize Cell Culture Exposed to Silver Nanoparticles.

The ever-increasing use of silver nanoparticles (nAg) in various products necessitates investigation of the behavior of biological systems encountering these particles. In this paper, considering maize as a biological model, the effects of colloidal nAg (<80nm) on its cell culture were investigated. For comparison purposes, silver nitrate was used as a representative of silver ion (Ag+). After stabilization of cell suspensions, they were treated with nAg and Ag+ (1 mg/l), then cell suspension growth was measured and the microscopic analysis and a cell viability test were performed. In addition, the activity of superoxide dismutase (SOD) enzyme was explored. Owing to the key role of retinoblastoma-related protein (RBR) in cell cycle as well as in development and differentiation processes, the relative expression of ZmRBR1 was studied in nAg and Ag+ exposure. Microscopic analyses revealed that cells in suspensions treated by nAg and Ag+ were morphologically classified into five types: embryogenic; larvae-like; long; swollen; and polarized. The results showed an increase in percentages of large and live cells in the treated suspensions. Remarkably, we observed some cells which were differentiated into trichomes along with some stages of trichome development in the treated cell suspensions. Moreover, exposure to nAg and Ag+ did not elevate the activity of SOD enzyme in the treated cells. Also, the relative expression of ZmRBR1 was slightly reduced in the treated cells. The findings of these experimentations indicated that nAg affected maize suspension-cultured cells in the same manner as Ag+.

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms.

In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Unveiling gene trait relationship by cross-platform meta-analysis on Chinese hamster ovary cell transcriptome.

In the past few years, transcriptome analysis has been increasingly employed to better understand the physiology of Chinese hamster ovary (CHO) cells at a global level. As more transcriptome data accumulated, meta-analysis on data sets collected from various sources can potentially provide better insights on common properties of those cells. Here, we performed meta-analysis on transcriptome data of different CHO cell lines obtained using NimbleGen or Affymetrix microarray platforms. Hierarchical clustering, non-negative matrix factorization (NMF) analysis, and principal component analysis (PCA) accordantly showed the samples were clustered into two groups: one consists of adherent cells in serum-containing medium, and the other suspension cells in serum-free medium. Genes that were differentially expressed between the two clusters were enriched in a few functional classes by Database for Annotation, Visualization, and Integrated Discovery (DAVID) of which many were common with the enriched gene sets identified by Gene Set Enrichment Analysis (GSEA), including extracellular matrix (ECM) receptor interaction, cell adhesion molecules (CAMs), and lipid related metabolism pathways. Despite the heterogeneous sources of the cell samples, the adherent and suspension growth characteristics and serum-supplementation appear to be a dominant feature in the transcriptome. The results demonstrated that meta-analysis of transcriptome could uncover features in combined data sets that individual data set might not reveal. As transcriptome data sets accumulate over time, meta-analysis will become even more revealing. Biotechnol. Bioeng. 2017;114: 1583-1592. © 2017 Wiley Periodicals, Inc.

Effect of some factors on the growth and regeneration of cell suspension of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.)

Ngoc Linh ginseng also known as Vietnamese ginseng (Panax Vietnamensis Ha et Grushv.) is a perennial medicinal plant. This plant is extremely rare and belongs to the Araliaceae family. Scientists are focusing on studies of Ngoc Linh ginseng nowadays. In this research, the effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 6-benzylaminopurine (BA), Kinetin (KIN), mineral salt formulations and cultural conditions, pH, sucrose concentration, medium volume on cell suspension culture of Panax vietnamensis Ha et Grushv. were investigated. In addition, growth curves and the effect of several plant grow regulators including a-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) on the regeneration of Ngoc Linh ginseng‘s cell suspension were also presented in this study. After 28 days in culture, the results showed that the best growth of a cell suspension of Ngoc Linh ginseng were obtained on ½MS liquid medium supplemented with 1.5 mg/l NAA, 50 g/l sucrose and the most suitable pH was 6.3. The acceptable medium volume for cell suspension growth was 30 ml. The growth curve of Ngoc Linh ginseng’s cell suspension showed that it should be subcultured at the beginning of the stationary phase approximately the 14th-16th day of culture. Ngoc Linh ginseng’s cell suspension exhibited the strongest growth at this time. When Ngoc Linh ginseng’s cell suspension was transferred to fresh medium, somatic embryos were formed in MS medium supplemented with 3.0 mg/l NAA after 30 days culture. The results shown that the potential regeneration of cell suspension of Ngoc Linh ginseng is very high.

82. Scalable, Animal-Free, Suspension-Based Production of Siv Lentiviral Vectors

We are developing lung gene transfer vectors to direct abundant, sustained transgene expression in three therapeutic compartments: (i) within lung cells to treat lung disorders such as cystic fibrosis (CF), (ii) secreted from lung cells into the lung lumen to treat viral infections and airway diseases such as alpha1 anti-trypsin deficiency, (iii) secreted from nasal and lung cells into the circulatory system to treat hematologic and a range of other disorders. Such diseases are associated with a high burden of care often involving frequent antibiotic treatments, physiotherapy and supportive systemic infusions; and considerable patient morbidity and mortality. We have developed an efficient lung gene transfer platform based on a European and US Pharmacopoeial compliant, minimal recombinant SIV lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN). Initially, production of rSIV.F/HN vectors was established in a serum-dependent, calcium phosphate-mediated, transient transfection process. Here we report the development of a scalable, animal-free, cGMP-compliant suspension cell culture based upstream production (USP) method. Serum-free media screening identified a suspension cell growth conditions that support efficient (>70%) multiple plasmid transient transfection of HEK293T cells with a range of cGMP-compliant liposomal and polymeric gene transfer agents. Small volume (40–400mL) studies in Erlenmeyer shake flasks and Design Of Experiments statistical approaches identified robust growth and transfection conditions that yield ≥5e6 TU/mL of unpurified rSIV. F/HN vectors. These parameters supported transition to 1L and 5L scale WAVE bioreactor cultures, where virus yield is maintained and plasmid DNA usage (a high proportion of the cGMP cost of goods) has been reduced to 0.33mg/L. F/HN pseudotype specific DNA removal and virus activation steps are incorporated into the downstream purification (DSP) method which relies on anion-exchange membrane virus capture and hollow-fibre UF/DF for vector purification and final formulation. In 56 independent, pH-controlled WAVE bioreactor cultures, purified yield exceeds 2.0e9 TU/L for a range of reporter gene constructs with a variety of promoters and 1.2e9 TU/L for a range of CFTR constructs (p>0.05; n=42/14 respectively). The final product directs abundant in vivo airway gene transfer, transducing 14.1% epithelial cells in the murine lung (p<0.001; 8e8 TU rSIV.F/HN hCEF EGFP), and directing CFTR transgene expression at ~30% of endogenous CFTR mRNA levels in the ovine lung (p<0.001; 8e8 TU rSIV.F/HN hCEF soCFTR2), exceeding presumed therapeutic levels. While these regulator friendly USP/DSP approaches have been tuned to yield highly purified and potent rSIV.F/HN vectors, the methodology is readily transferrable to VSV-G pseudotype production.

Growth Improvement of Nicotiana and Arabidopsis In Vitro by Microalgal Conditioned Media

Abstract Conditioned medium (CM) is a general term describing media in which cells have already been cultivated for some time. Such media, usually clarified by filtration, have been used by plant biotechnologists as additives sup-porting the growth of cell suspensions, organs and whole plants. This study examined the effect of CM obtained from green alga Desmodesmus subspicatus on the growth and functioning of the photosynthetic apparatus of Nicotiana tabacum and Arabidopsis thaliana in culture in vitro. Plants where cultured on CM diluted 1.25-, 2-and 5-fold with MS medium. The increase in fresh and dry weight was highest in tobacco and Arabidopsis cultured on CM/2 and CM/1.25 media. Those two concentrations also increased the amount of chlorophylls in both plants tested. CM improved parameter PI (reflecting the photosynthetic “vitality” of the organism) and electron transport efficiency, and increased the fraction of active reaction centers. Analysis of chlorophyll fluorescence in vivo suggests that the improvement of these plants grown in the presence of algal CM may result from stimulation of photosynthesis. Algal CM offers a convenient, cheap, universal supplement for stimulating the growth of higher plants in vitro.

Эффективность различных методов выращивания вируса африканской чумы свиней в гемопоэтических клетках

For African swine fever virus (ASFV) reproduction little publications are available, in particular, those concerning cultivation technologies. Moreover, the range of effective cell systems for cultivation is limited too. This report highlights the development and comparative evaluation of methods for African swine fever virus (ASFV) growth in porcine bone marrow cell (PBM) and leukocyte (PL) cultures for their efficacy. We showed the possibility of using cell suspension prepared from porcine bone marrow after its 24-hour storage at 4 to 6 °С for ASFV culture. The virus grown in the PBM cell suspension after the abovementioned storage period for porcine bone marrow had expired did not reduce its infectious activity, and accumulated with titers of 7.25 to 7.50 lg TCID 50/cm 3. The conditions for ASF virus culture in hematopoietic cells using stationary devices like KASM (a cassette unit for monolayer culture), a technique for roll-bottle culture in a circular monolayer or in a cell suspension, and biological reactors (fermentors) for a suspension culture were analyzed. When using these culture methods, ASF virus steadily accumulated in the PBM or PL cells with high titers ranging from 7.25 to 8.00 lg HAU 50/cm 3. Nevertheless, ASF virus growth in hematopoietic cell suspensions using automated reactors was found to be the most effective, technologically advanced and productive technique. In this case, the virus titers were as high as 7.50 to 8.00 lg HAU 50/cm 3. Analyzing the results of the studies we concluded that the above methods were more efficient as compared to ASFV growth in a stationary cell culture using flasks, and could be used for large scale cultivation of this biological agent in vaccinology and diagnostics of African swine fever infection.

Fungal endophytes enhanced the growth and production kinetics of Vinca minor hairy roots and cell suspensions grown in bioreactor

A vincamine positive hairy root clone of Vinca minor with integration of all the three- rolA, rolB and rolC genes and its subsequently raised cell suspensions were treated with culture filtrate of four endophytic fungi namely Chaetomium globosum; Aspergillus niveoglaucus; Paecilomyces lilacinus and Trichoderma harzianum. Addition of 10 % v/v T. harzianum culture filtrate boosted maximum biomass accumulation and highest total alkaloid content (TAC) both in the hairy roots and in the cell suspensions. Treated (10 % v/v T. harzianum) hairy roots registered growth index (GI) of 640 ± 28.3 and TAC of 3.6 ± 0.1 dry wt% in comparison to non-treated roots (GI = 420 ± 14.2; TAC = 3.1 ± 0.3 dry wt%). Treated cell suspensions showed GI of 600 ± 19.1 and TAC of 1.67 ± 0.03 dry wt% in comparison to non-treated cell suspensions (GI = 250 ± 12.6; TAC = 1.15 ± 0.02 dry wt%). The hairy roots and the cell suspensions were successfully up-scaled in the 5 l stirred tank bioreactor with respective GI of 850.0 and 654.0 under optimized conditions. On Real time (qPCR) analysis, treated hairy roots showed fourfold to sixfold enhanced tryptophan decarboxylase (TDC) transcript level [relative quantity value (RQ) = 4.64 ± 0.30 (shake flask); RQ = 5.95 ± 0.31 (bioreactor)] while treated cell suspensions showed only two fold increase in TDC transcript [RQ = 2.1 ± 0.26 (shake flask); RQ = 2.5 ± 0.21 (bioreactor)]. Similarly, fivefold to sixfold [RQ = 5.6 ± 0.20 (shake flask); RQ = 6.7 ± 0.49 (bioreactor)] and threefold to fourfold [RQ = 3.5 ± 0.18 (shake flask); RQ = 3.8 ± 0.68 (bioreactor)] increased transcript of strictosidine synthase (STR) in treated hairy roots and cell suspensions, respectively was observed. Treated shake flask roots showed 0.002 dry wt% vincamine that was further enhanced in the bioreactor grown treated roots (0.015 dry wt%). No vincamine was detected in the shake flask culture or in the bioreactor grown cell suspensions.

Serum-free medium optimization based on trial design and support vector regression.

The Plackett-Burman design and support vector machine (SVM) were reported to be used on many fields such as some feature selections, protein structure prediction, or forecasting of other situations. Here, with suspension adapted Chinese hamster ovary (CHO) cells as the object of study, a serum-free medium for the culture of CHO cells in suspension was optimized by this method. Support vector machine based on genetic algorithm was used to predict the growth rate of CHO and prove the results from the trial designs. Experimental results indicated that ZnSO4, transferrin, and bovine serum albumin (BSA) were important ones. The same conclusion was arrived at when the support vector regression model analyzed the experimental results. With the methods mentioned, the influence of 7 medium supplements on the growth of CHO cells in suspension was evaluated efficiently.

Antitumor activity of the dietary diterpene carnosol against a panel of human cancer cell lines.

Dietary phytochemicals found in vegetables and fruits consist of a wide variety of biologically active compounds with anti-carcinogenic activity. The aim of this study was to evaluate the antigrowth activity of carnosol, a dietary diterpene, as a single agent or in combination with other dietary phytochemicals or chemotherapeutic drugs against a panel of tumor cell lines. Carnosol decreased cell viability in human breast, ovarian, and intestinal tumor cell lines, and inhibited cancer cell adhesion on fibronectin and growth of cancer cells in suspension. Carnosol also inhibited EGF-induced epithelial mesenchymal transition in ovarian cancer cells. The combination treatment with other dietary phytochemicals increased the anti-proliferative activity of carnosol. The combination with curcumin resulted in a synergistic reduction of vitality in SKOV-3 and MDA-231 cells and potently inhibited viability of primary cancer cells isolated from the pleural fluid or ascites of patients with metastatic cancers. These results provide additional evidence about the anticancer role of carnosol and its potential in blocking the growth of tumor cells.