Effect of shear stress on anthraquinones production by Rubia tinctorum suspension cultures

Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon.     

Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

Design of bioreactors suitable for plant cell and tissue cultures

Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In contrast to traditionally grown “whole wild plants” or “whole transgenic plants”, their production in bioreactors guarantees defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing acceptance over the last 10 years. Plants for human health in the post-genome era, PSE congress 26.8.2007–29.8.2007, Helsinki.     

Anthraquinones in cell suspension cultures of Morinda citrifolia

From cell suspension cultures of Morinda citrifolia five known anthraquinones, rubiadin, lucidin, morindone, lucidin-3-β-primeveroside and morindone-6-β-primeveroside, and seven new anthraquinones were isolated. Six of the seven new quinones were characterized as 2-methyl-3,5,6-trihydroxyanthraquinone, 3-hydroxymorindone, 5,6-dihydroxylucidin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, 3-hydroxymorindone-6-β-primeveroside and 5,6-dihydroxylucidin-3-β-primeveroside, respectively.     

Immobilization of Rubia tinctorum L. suspension cultures and its effects on alizarin and purpurin accumulation and biomass production

The present study is investigating the immobilization of Rubia tinctorum L. suspension cultures. The effects of three inoculation volumes and three immobilization materials (loofa, sisal and jute) on fresh and dry weights of biomass as well as on alizarin and purpurin production were determined in this study. Two grams of four-week old callus tissue were transferred to liquid medium to establish suspension cultures. After four weeks, suspension cultures of R. tinctorum at concentration of 8?×?10⁵?living cells/ml were immobilized with lignocellulosic materials and the cells were attached to all immobilization materials at the end of the first week and started to form aggregates on them. At the fourth week of these batch systems, biomass was measured approximately three times higher than the starting suspension cultures. The highest fresh weight was obtained (339.40?g/l) from sisal with ? inoculation ratio. Immobilization materials and inoculation volumes had an effect on dry weights, and accordingly, the most effective combinations were jute with ? (J3) and ? (J1) inoculation volumes with 7.86 and 7.82?g/l dry weights, respectively. Alizarin and purpurin contents of immobilized cells, analyzed with U-HPLC method, were 6.05 and 22.91 times higher than inoculated cells. All immobilization materials used in this study had no negative effect on to cells and biomass accumulation was enhanced. Concomitantly with rapid biomass increase, alizarin and purpurin production was ascended.     

Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm³ of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm⁻³ indolebutyric acid, 0.5 mg dm⁻³ kinetin and 30 g dm⁻³ sucrose. A 1 % CO₂ supply was found beneficial for the production of cell mass; however, increasing CO₂ concentration to 2.5 and 5 % decreased the biomass accumulation. CO₂ enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO₂ supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.     

Anthraquinones production,hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H₂O₂) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l^–1, intracellular AQ content at 42 mg g^–1 DW, and H₂O₂ level at 9 mol g^–1 FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g^–1 FW and total carotenoids at 13.3 mg g^–1 FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H₂O₂ levels. Vitamin C content at 30–120 g g^–1 FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400–500 g g^–1 FW in 7-day-old cultures from all medium strategies and reduced to 50–150 g g^–1 FW on day 14 and 21; as compared to 60 g g^–1 FW in callus and 200 g g^–1 FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.Abbreviations AQ Anthraquinone - DW Dry cell weight - FW Fresh cell weight - G Intermediary medium - M Maintenance medium - MDA Malondialdehyde - P Production medium - ROS Reactive oxygen species - TBA Thiobarbituric acid - t_d Doubling time     

Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus

Cell cultures of Catharanthus roseus were scaled up to volumes of 50001 using conventional reactors equipped with flat-blade impellers. The behavior of the fermenter grown cells was compared with corresponding shake flask experiments with respect to growth and indole alkaloid inducibility and production. The limits and problems of transferring shake flask experiments of culture systems such as Catharanthus, in which alkaloid production depends greatly upon the physiological state of the cells, to large scale multistage processes is discussed.     

Synergistic effect of methyl jasmonate and cyclodextrins on anthraquinone accumulation in cell suspension cultures of Morinda citrifolia and Rubia tinctorum

Plant in vitro culture is a platform for producing secondary metabolites that combines safety, quality and low environmental impact. Besides, it is possible to increase the accumulation of these compounds by different strategies, such as elicitation. In this work, we analyzed the effects of the combination of methyl jasmonate (MeJ) and two cyclodextrins (CDs) on the production of anthraquinones (AQs) in cell cultures of Rubiaceae (Morinda citrifolia and Rubia tinctorum). These secondary metabolites have been traditionally used as dyes and have interesting therapeutic applications. The experiments were designed according to a full factorial design of two factors (MeJ and a CD) in two levels (0 and 0.1 mM for MeJ, and 0 and 20 mM of the CD). MeJ and (2-hydroxypropyl)-β-cyclodextrin (HPCD) synergistically increased intracellular AQ content in suspension cultures of R. tinctorum, and, to a lesser extent, in suspension cultures of M. citrifolia. Combination of MeJ with another CD, 2-methyl-β-cyclodextrin, led to a more intense and later increase in AQ content in cell cultures of R. tinctorum when compared to MeJ–HPCD treatment. However, the combination of CD and MeJ failed to induce a drastic AQ release to the culture media. Nevertheless, our results show that combination of strategies (using a CD and MeJ) was successful to increase secondary metabolite accumulation in suspension cultures. To our knowledge, this is the first report of synergistic effect of MeJ and CD on AQ accumulation in plant in vitro cultures.

     

Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures

“Molecular farming” in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative “factory”. However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.     

Stereoselective reduction of ketones by plant cell cultures

Eighteen species of plant cell could reduce of 2-pentanone, acetophenone and ethyl acetoacetate to the S-alcohols with yields ranging from 20 to 100 % (w/w) and optical purity ranging from 65 to 99 % enantiomeric excess (e.e.). © Rapid Science Ltd. 1998     

Methyl jasmonate enhanced production of rosmarinic acid in cell cultures of Satureja khuzistanica in a bioreactor

The growing interest in rosmarinic acid (RA), an ester of caffeic acid and 3,4‐dihydroxyphenyl lactic acid, is due to its biological activities, which include cognitive‐enhancing effects, slowing the development of Alzheimer's disease, cancer chemoprotection, and anti‐inflammatory activity. Inspired by the challenge of meeting the growing demand for this plant secondary metabolite, we developed a biotechnological platform based on cell suspension cultures of Satureja khuzistanica. The high amounts of RA produced by this system accumulated mainly inside the cells. To further improve production, two elicitors, 100 μM methyl jasmonate (MeJA) and 40 mM cyclodextrin (CD), were tested, separately and together. MeJA increased RA productivity more than 3‐fold, the elicited cultures achieving an RA production of 3.9 g L^?1 without affecting biomass productivity. CD did not have a clear effect on RA production, and under the combined treatment of MeJA + CD only a small amount of RA was released to the medium. When the cell culture was transferred from a shake flask to a wave‐mixed bioreactor, a maximum RA production of 3.1 g L^?1 and biomass productivity of 18.7 g L^?1 d^?1 was achieved under MeJA elicitation, demonstrating the suitability of S. khuzistanica cell suspensions for the biotechnological production of this bioactive plant secondary metabolite.     

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone‐induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone‐triggered ABA generation and assayed the effect of fluridone on ozone‐induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone‐triggered ABA generation but also blocked the ozone‐induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose‐dependent. Interestingly, the suppression of fluridone on ozone‐induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone‐induced Taxol production of T. chinensis suspension cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Characterization of buffelgrass (Cenchrus Ciliaris L.) cell suspension cultures

Summary Cell suspension cultures of buffelgrass were established from two types of callus, a friable tan callus and a brown gelatinous callus, using Murashige and Skoog medium containing 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The friable callus formed a rapidly growing suspension culture, designated BG, which had a doubling time of 2.5 days. The gelatinous callus formed a very slow-growing suspension culture, designated BGG, which had a doubling time of 1 mo. During growth, the medium of the BGG line slowly increased in viscosity, becoming a thickened gel by the end of the subculture period. Both lines had high cell viability. Embryogenesis could be induced in both lines by culturing on charcoal-containing, 2,4-D-free medium. No embryos formed in the absence of charcoal.     

Biotransformation of podophyllotoxin by Hordeum vulgare cell suspension cultures

Hordeum vulgare cell suspension cultures were used to modify podophyllotoxin (1) One major product (1a) and one minor product (1b) were detected in both the culture medium and cells. To optimize the yield of compound 1a, we showed that: (1) the optimal concentration of added podophyllotoxin (1) was 33 mg L^-1; higher concentrations caused cell toxicity; (2) the stage of the cell cycle (lag/log/stationary) at which podophyllotoxin was added only marginally affected the yield of compound 1a; the optimal addition time was after lag phase, in which the yield of compound 1a reached ca. 76%, and (3) biotransformation of podophyllotoxin (1) was relatively slow; podophyllotoxin fed at 4 days after subculture resulted in yields of compound 1a of ca. 56, 64 and 76% after an additional 3, 6 and 10 days of incubation, respectively. Product 1a was purified and identified as isopicropodophyllone (1a) based on MS and NMR data.     

Cultivation of Arabidopsis cell cultures in a stirred bioreactor at variable oxygen levels: Influence on tocopherol production

Abstract

In plants, an increased production of toxic oxygen species is commonly observed under low oxygen stress, but cellular responses still have to be fully investigated. Plant cell cultures can be a valuable tool to study plant metabolic responses to various environmental stresses including low oxygen condition. Arabidopsis suspension cultures growing in shake flasks were subjected to hypoxia by stopping shaking for different intervals, showing an increase of the antioxidant metabolite α‐tocopherol. In order to obtain a more controlled condition, cultivation of Arabidopsis suspension cultures was established in a 5‐l stirred bioreactor. A constant aeration of 20% dissolved oxygen was found to be the most suitable for cell growth. A 4‐h anoxic shock was induced by suspending the aeration and flushing into the vessel with nitrogen. During the anoxic stress, tocopherol levels resulted increased at the end of the treatment, indicating that the complete oxygen deprivation, indeed, induced a defence response involving antioxidant metabolism. The presence of an oxidative stress as a consequence of anoxic condition was also confirmed by the increased levels of H₂O₂. Overall, these results indicate that Arabidopsis suspension cultures grown in a stirred bioreactor can be a useful in vitro system for investigating low oxygen stress.     

Non‐Newtonian rheology in suspension cell cultures significantly impacts bioreactor shear stress quantification

The fields of regenerative medicine and tissue engineering require large‐scale manufacturing of stem cells for both therapy and recombinant protein production, which is often achieved by culturing cells in stirred suspension bioreactors. The rheology of cell suspensions cultured in stirred suspension bioreactors is critical to cell growth and protein production, as elevated exposure to shear stress has been linked to changes in growth kinetics and genetic expression for many common cell types. Currently, little is understood on the rheology of cell suspensions cultured in stirred suspension bioreactors. In this study, we present the impact of three common cell culture parameters, serum content, cell presence, and culture age, on the rheology of a model cell line cultured in stirred suspension bioreactors. The results reveal that cultures containing cells, serum, or combinations thereof are highly shear thinning, whereas conditioned and unconditioned culture medium without serum are both Newtonian. Non‐Newtonian viscosity was modeled using a Sisko model, which provided insight on structural mechanisms driving the rheological behavior of these cell suspensions. A comparison of shear stress estimated by using Newtonian and Sisko relationships demonstrated that assuming Newtonian viscosity underpredicts both mean and maximum shear stress in stirred suspension bioreactors. Non‐Newtonian viscosity models reported maximum shear stresses exceeding those required to induce changes in genetic expression in common cell types, whereas Newtonian models did not. These findings indicate that traditional shear stress quantification of cell or serum suspensions is inadequate and that shear stress quantification methods based on non‐Newtonian viscosity must be developed to accurately quantify shear stress.