Shear stress effects on plant cell suspension cultures in a rotating wall vessel bioreactor

Article (PDF Available)inJournal of Industrial Microbiology and Biotechnology 22(1):44-47 · January 1999with102 Reads
DOI: 10.1038/sj.jim.2900600
A rotating wall vessel, designed for growth of mammalian cells under microgravity, was used to study shear effects on Taxus cuspidata plant suspension cell cultures. Shear stress, as quantified by defined shear fields of Couette viscometers, improved specific cell growth rates and was detrimental to volumetric product formation rates.
    • Moreover, the plant contains high contents of phenolics and flavonoids, indicating that these compounds contribute to its antiradical and antioxidant activity [2] . Plant cell suspension cultures have the potential for largescale secondary metabolites production [9] and act as attractive entities for the production of biologically active compounds [10]. They are most often heterotrophic and, therefore, dependent upon the exogenous supply of carbon for growth.
    [Show abstract] [Hide abstract] ABSTRACT: Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9 %). Moreover, the effects of 3 % repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5 %. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7 % were optimum for both classes of metabolites, respectively. Following 3 % extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.
    Article · Aug 2016
    • sativa L.) 3–12 mg/L/day for individual cycle; overall productivities up to 7.7 mg/L/day. the subject of extensive investigation (Camacho et al., 2000; Namdev and Dunlop, 1995; Sowana et al., 2001; Sun and Linden, 1999; Zhong et al., 2009 Zhong et al., , 1994) however, the precise mechanism by which shear stress induces cell damage is not well-understood and has been very difficult to study due to the incredible diversity of plant cell lines, the varying aggregate size distribution and their assorted cell morphologies (). As such, susceptibility of plant cells to shearing forces varies widely among different cell lines.
    [Show abstract] [Hide abstract] ABSTRACT: "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.
    Full-text · Article · May 2011
    • Plant secondary metabolism is an important source of fine chemicals such as drugs, dyes, flavors, and fragrances. The use of plant in vitro culture techniques to produce these chemicals is increasing (Verpoorte et al., 2000) and the following examples can be mentioned: benzophenanthridine alkaloids by Haider et al. (1997); anthocyanin, anthocyanidin, and carotenoids by Shibli et al. (1997); ginseng saponin by Zhong et al. (1997); flavonoids by Fang et al. (1998); daidzein and coumestrol by Bourgaud et al. (1999); verbascoside by Nezbedová et al. (1999); taxol by Sun and Linden (1999); shikonin by Boehm et al. (2000); furano-coumarins by Massot et al. (2000); anthocyanin by Miyanaga et al. (2000); coumarin and emetine by Pereira et al. (2000) and Bais et al. (2001); artemisinin and stigmasterol by Xie et al. (2000); and rosmarinic acid by Kim et al. (2001). Plant cysteine proteases (papain, chrymopapain, caricain, papaya proteinase IV, actinidin, bromelains, ananain, comasain) are among the extensive list of useful secondary metabolites.
    [Show abstract] [Hide abstract] ABSTRACT: Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.
    Full-text · Article · May 2003
    • The use of a clinostat for studying how gravity affects plant growth dates back well before the advent of space flight, possibly to the early 1700's (Cogili, 1992; Gruener, no date). Various review articles along with a growing number of primary research papers are available ρ ρ ρ in the more recent literature which describe the experimental merits and physical constraints, predominantly concerning plant and suspension cell culture studies, of using a clinostat (Brown et al., 1996; Driss-Ecole et al., 1994; Duke et al., 1998; Hashemi et al., 1999; Laurinavicius et al., 1994; Sarkar et al., 2000; Schnabl et al., 1996; Sievers and Heinowicz, 1992; Smith et al., 1999; Wolf et al., 1993) or the RWV bioreactor (Fang et al., 1997a; Fang et al., 1997b; Fang et al., 1997c; Fang et al., 2000; Hammond et al., 1999; Klement and Spooner, 1993; Lewis et al., 1993; Sun and Linden, 1999; Tsao et al., 1992). Empirical results obtained using a clinostat or rotating wall vessel are frequently, though not always, found to be similar to those observed in comparable space flown experiments.
    [Show abstract] [Hide abstract] ABSTRACT: The environment created on Earth within a clinostat or Rotating Wall Vessel (RWV) bioreactor is often referred to as "simulated microgravity". Both devices utilize constant reorientation to effectively nullify cumulative sedimentation of particles. Neither, however, can fully reproduce the concurrent lack of structural deformation, displacement of intercellular components and/or reduced mass transfer in the extracellular fluid that occur in actual weightlessness. Parameters including density, viscosity, and even container geometry must each be considered to determine the overall gravity-dependent effects produced by either a clinostat or the RWV bioreactor; in addition, the intended application of these two devices differs considerably. A state of particle "motionlessness" relative to the surrounding bulk fluid, which is nearly analogous to the extracellular environment encountered under weightless conditions, can theoretically be achieved through clinorotation. The RWV bioreactor, on the other hand, while similarly maintaining cells in suspension as they continually "fall" through the medium under 1 g conditions, can also purposefully induce a perfusion of nutrients to and waste from the culture. A clinostat, therefore, is typically used in an attempt to reproduce the quiescent, unstirred fluid conditions achievable on orbit; while the RWV bioreactor ideally creates a low shear, but necessarily mixed, fluid environment that is optimized for suspension culture and tissue growth. Other techniques for exploring altered inertial environments, such as freefall, neutral buoyancy and electromagnetic levitation, can also provide unique insight into how gravity affects biological systems. Ultimately, all underlying biophysical principles thought to give rise to gravity-dependent physiological responses must be identified and thoroughly examined in order to accurately interpret data from flight experiments or ground-based microgravity analogs.
    Article · Jul 2001
    • Shear stress may affect plant cell growth and cellular metabolism negatively or positively, depending on the level of applied shear stress, the properties of the cell line, and its physiological state (Dunlop et al., 1994). Shear stress at 2.1 s -1 was found positive to paclitaxel production, compared to zero shear stress using T. cuspidata (Sun and Linden, 1999); however, greater shear stress levels resulted in physiological changes of the cells that, at the extreme, led to cell death. From the comparison of cultures in the spinner flasks, growth yield (Y x/s ) in the semicontinuous culture with cell recycle was less than 50% of the batch culture.
    [Show abstract] [Hide abstract] ABSTRACT: Suspension cultures of Taxus canadensis were elicited with methyl jasmonate (MJ) under defined headspace ethylene concentrations. Kinetic studies of growth, nutrient consumption, pH variation, and paclitaxel accumulation were conducted in batch cultures and semicontinuous culture with total cell recycle. A dramatic increase of paclitaxel was obtained when the cultures were elicited with 100 microM MJ, but cell growth was thereby arrested. Supplementation of acetyl-CoA and MJ to the culture proved to be another way to improve paclitaxel yields. Using semicontinuous culture with total cell recycle, paclitaxel accumulation was increased by a factor of 4.0 relative to that in the batch culture during 35 days of cultivation.
    Full-text · Article · Dec 1999
    • The results are in agreement with the previous findings suggesting that plant cells are most sensitive to hydrodynamic shear stress in the range of shear rate from 30 to 300 s -1 (Sun & Linden 1999). No suspensor cells form at shear rate of 140 s -1 and suspensor cells form under lower shear rates of 86, 29, 14 and 9 s -1 .
    [Show abstract] [Hide abstract] ABSTRACT: The effect of steady shear stress on somatic embryos were investigated in a flow chamber and evaluated at different time intervals using microscopy technique. The development of meristematic cell clusters, i.e. the immature embryos, into a polarized somatic embryo, and the effect on the localization of the suspensor cells that form during development of the immature embryos, were studied as a function of shear stresses. With the distribution and growth rate of the meristematic and suspensor cells, the effect of stress on the embryo development was established. Furthermore, the effect of shear stress on the cells at molecular level, the reaction of integrin-like proteins, the production of reactive oxygen species and the pore size of the cell walls involved in the shear stress responses, were investigated with molecular techniques. In general, shear stress inhibits meristematic cells growth. Meristematic cells grow fastest at shear rate of 86 s-1 among all the tested shear stress conditions. By combining the results of meristematic cells growth and suspensor cells formation, it suggests that there is a critical shear rate between 86 and 140 s-1, at which no suspensor cells form. The unidirectional flow with different shear stresses helps the polarized growth and the unidirectional alignment of suspensor cells. Reactive oxygen species and integrin-like protein are detected in the stressed cells as cellular responses to shear stresses. By monitoring the pore size and uptake time of cells to macromolecules with solute-exclusive experiments, it suggests that the stressed cells expedite the response to plasmolyzing components that are used to induce maturation treatment thus affect the response to maturation stimuli.
    Article · Biotechnology Progress
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