Continuous production of glycosides by a bioreactor using ginseng hairy root culture

Ginseng (Panax ginseng) hairy-root culture, established by transformation with the Ri plasmid of Agrobacterium rhizogenes, had a higher potential to biotransform (RS)-2-phenylpropionic acid (PPA) to (RS)-2-phenylpropionyl -d-glucopyranoside (1) (71% conversion ratio), (2RS)-2-O-(2-phenylpropionyl)-d-glucose (2) (8%), (2S)-2-phenylpropionyl 6-O--d-xylopyranosyl--d-glucopyranoside (3) (10%) and a myo-inositol ester of (R)-2-phenylpropionic acid (4) (5%). Moreover, the hairy root excreted about a half of the conversion products, 46.8%. The continuous glycosylation of PPA was carried out using a bioreactor with ginseng hairy root, and the continuous long-term reaction for 2 months was successfully made at a high conversion ratio, 30% or more on average.This work is Part 84 in the series Studies on Plant Tissue Culture. For Part 83, see Asaka et al. (1993) Correspondence to: T. Furuya     

Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis

ABSTRACT

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.     

Vitis vinifera culture in a non-conventional bioreactor: the reciprocating plate bioreactor

This paper is concerned with the potential use of a reciprocating plate bioreactor (RPB) for suspended plant cell cultures. The agitation mechanism of the RPB system, a plate stack, was first evaluated in pure water and in pseudocells medium of 20, 40 and 60% of PCV. As the pseudocell concentration increases, the oxygen mass transfer coefficient, K_La, significantly decreases. Correlations were established for each plate stack and concentration with good prediction of K_La. Three fermentations were performed with Vitis vinifera cells, two in the RPB system and one in shake flasks. Shake flask cultures showed better performance whereas the first fermentation performed with the RPB showed the lowest performance. The lower growth observed was attributed to the operating conditions for aeration and the dissolved oxygen control strategy. CO₂ stripping in the initial portion of the fermentation led to lower biomass growth. The second fermentation, with more appropriate operating conditions, appears to follow the trend of shake flask cultures but was terminated after 5 days due to contamination. The RPB has the potential to be used for suspended plant cell cultures but significant research needs to be performed to find optimal operating conditions but, more importantly, to make appropriate modifications to ensure the sterility of the bioreactor over long time periods.     

Effects of memory on spatial heterogeneity in neutrally transmitted culture

We explore how cultural heterogeneity evolves without strong selection pressure or environmental differences between groups. Using a neutral transmission model with an isolation-by-distance spatiality, we test the effect of a simple representation of cultural ‘memory’ on the dynamics of heterogeneity. We find that memory magnifies the effect of affinity while decreasing the effect of individual learning on cultural heterogeneity. This indicates that, while the cost of individual learning governs the frequency of individual learning, memory is important in governing its effect.     

Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture

Three-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired.     

Continuous production of scopolamine by a culture of Duboisia leichhardtii hairy root clone in a bioreactor system

The scopolamine-releasing hairy root clone DL47-1 of Duboisia leichhardtii was cultured in an Amberlite XAD-2 column-combined bioreactor system for continuous production of scopolamine. The medium used was continuously exchanged during culture to maintain the electrical conductivity of the medium constant. After culturing the hairy roots in the system for 11 weeks, 0.5 g/l of scopolamine was obtained in the column. When the roots were cultures in the reactor system containing polyurethane foam or stainless-steel mesh to support the hairy roots, scopolamine recovery was increased. Thereafter, a two-stage culture, the first stage in the medium for hairy root growth and the second stage in the medium for scopolamine release, was carried out in this system by using a turbine-blade reactor with stainless-steel mesh as a support. Under these conditions, 1.3 g/l of scopolamine was recovered during 11 weeks of culture in the medium for scopolamine release. This bioreactor system seems applicable for the production of various plant metabolites by cultures of hairy roots.Correspondence to: T. Muranaka     

Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes

Agrobacterium rhizogenes strain 15834, which incites hairy root disease in plants, harbors three large plasmids: pAr15834a (107 x 10(6) daltons), pAr15834b (154 x 10(6) daltons), and pAr15834c (258 x 10(6) daltons). Kanamycin-resistant transconjugants were selected in a cross of kanamycin-resistant derivate of strain 15834 and an avirulent recipient. The transconjugants belonging to one class were virulent and contained all three donor plasmids. These transconjugants also acquired sensitivity to the bacteriocin agrocin 84. The loss of plasmids from virulent transconjugants during growth at 37 degrees C indicated that virulence genes reside on pAr15834b, whereas agrocin 84 sensitivity genes reside on pAr15834a. The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain of A. rhizogenes. Restriction endonuclease fragment analysis of plasmids from the transconjugants and the donor revealed that pAr15834c is a cointegrate of pAr15834a and pAr15834b. Kanamycin-resistant transconjugants belonging to a second class were avirulent and contained an altered form of pAr15834b. Strain 15834 can utilize octopine. However, this trait was not detected in any of the transconjugants. Octopine is not synthesized by infected plant tissue.     

Hairy root research: recent scenario and exciting prospects

High stability of the production of secondary metabolites is an interesting characteristic of hairy root cultures. For 25 years, hairy roots have been investigated as a biological system for the production of valuable compounds from medicinal plants. A better understanding of the molecular mechanism of hairy root development, which is based on the transfer of Agrobacterium rhizogenes T-DNA into the plant genome, has facilitated its increasing use in metabolic engineering. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for the pharmaceutical industry. In addition, hairy roots offer promise for phytoremediation because of their abundant neoplastic root proliferation. Recent progress in the scaling-up of hairy root cultures is making this system an attractive tool for industrial processes.     

Incomplete mixing effects in a continuous culture laboratory-scale bioreactor

A laboratory bioreactor (1 L working volume) operating in chemostat mode was utilised for the culture of the polymorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 undergoing fermentation of cheese whey permeate. Analysis of kinetic data revealed that the experimentally measured lactose concentration profiles in the exit line from the chemostat varied when the inlet feed concentrations ranged from 5–20 g lactose L–1. Further analysis revealed that this was due to about 3.5% of the feed bypassing the mixing zone. This incomplete mixing occurred despite an agitation rate of 800 rpm, an aeration rate of 1 vvm and the presence of internal baffles in the bioreactor. Without allowing for incomplete mixing, the estimation of the apparent Ks value for the organism could be out by a factor of five, over the range of feed concentrations employed. © Rapid Science Ltd. 1998     

Computational fluid dynamics modeling of mass-transfer behavior in a bioreactor for hairy root culture. II. Analysis of ultrasound-intensified process

Recently, cichoric acid production from hairy roots of Echinacea purpurea was significantly improved by ultrasound stimulation in an airlift bioreactor. In this article, the possible mechanism on ultrasound-intensified hairy root culture of E. purpurea in the bioreactor was elucidated with the help of computational fluid dynamics (CFD) simulation, membrane permeability detection, dissolved oxygen concentration detection, confocal laser-scanning microscopy (LSM) observation, and phenylalanine ammonium lyase (PAL) activity analysis. The CFD model developed in Part I was used to simulate the hydrodynamics and oxygen mass transfer in hairy root bioreactor culture stimulated by ultrasound. A dynamic mesh model combined with a changing Schmidt number method was used for the simulation of the ultrasound field. Simulation results and experimental data illustrated that ultrasound intensified oxygen mass transfer in the hairy root clump, which subsequently stimulated root growth and cichoric acid biosynthesis. Ultrasound increased the hairy root membrane permeability, and a high root membrane permeability of 0.359 h(-1) was observed at the bottom region in the bioreactor. LSM observation showed that the change in the membrane permeability recovered to normal in the further culture after ultrasound stimulation. PAL activity in the hairy roots was stimulated by ultrasound increase and was correlated well to cichoric acid accumulation in the hairy roots of E. purpurea.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Design and characterization of a new bioreactor for continuous ultra-slow uniaxial distraction of a three-dimensional scaffold-free stem cell culture

A computer controlled dynamic bioreactor for continuous ultra-slow uniaxial distraction of a scaffold-free three-dimensional (3D) mesenchymal stem cell pellet culture was designed to investigate the influence of stepless tensile strain on behavior of distinct primary cells like osteoblasts, chondroblasts, or stem cells without the influence of an artificial culture matrix. The main advantages of this device include the following capabilities: (1) Application of uniaxial ultra-slow stepless distraction within a range of 0.5-250 μm/h and real-time control of the distraction distance with high accuracy (mean error -3.4%); (2) tension strain can be applied on a 3D cell culture within a standard CO(2) -incubator without use of an artificial culture matrix; (3) possibility of histological investigation without loss of distraction; (4) feasibility of molecular analysis on RNA and protein level. This is the first report on a distraction device capable of applying continuous tensile strain to a scaffold-free 3D cell culture within physiological ranges of motion comparable to distraction ostegenesis in vivo. We expect the newly designed microdistraction device to increase our understanding on the regulatory mechanisms of mechanical strains on the metabolism of stem cells.     

The nature of zooplankton spatial heterogeneity in a nonriverine impoundment

Knowledge of spatial heterogeneity characteristic of reservoir plankton communities is fundamental to a variety of ecological studies. Degree of spatial heterogeneity in the zooplankton community of Center Hill Reservoir, with water residence times of 50–250 days, was positively correlated with rate of water influx. Important spatial differences resulted from the contrast between zooplankton associated with new and longer-impounded water. The nature of spatial heterogeneity differed fundamentally from the more riverine impoundments where spatial differences are often persistent and characterized by gradual change (as opposed to contrast) in plankton assemblages with respect to location. Magnitude of plankton spatial heterogeneity in nonriverine impoundments may be predictable from inflow rates. Areas, between which major differences in plankton communities exist, may also be definable from knowledge of inflow dispersal patterns in these impoundments.     

Pilot-scale culture of adventitious roots of ginseng in a bioreactor system

A pilot-scale culture of multiple adventitious roots of ginseng was established using a balloon-type bubble bioreactor. Adventitious roots (2 cm) induced from callus were cultured in plastic Petri dishes having 20 ml of solid Schenk and Hildebrandt (1972) medium containing 3% sucrose, 0.15% gelrite, and 24.6 μM indole-3-butric acid. An average of 29 secondary multiple adventitious roots were produced after 4 weeks of culture. These secondary roots were elongated on the same medium, reaching a length of 5 cm after 6 weeks of culture. A time course study revealed that maximum yields in 5-l and 20-l bioreactors were approximately 500 g and 2.2 kg at day 42 with 60 g and 240 g inoculations, respectively. Cutting twice during the culture increased the total amount of biomass produced. The root biomass in a 20-l balloon-type bubble bioreactor was 2.8 kg at harvest with 240 g of inoculum after 8 weeks of culture. The total saponin content obtained from small-scale and pilot-scale balloon type bubble bioreactors was around 1% based on dry weight. Inoculation of 500 g fresh weight of multiple adventitious roots into a 500 l balloon-type bubble bioreactor with cutting at 4 and 6 weeks after inoculation produced approximately 74.8 kg of multiple roots. The ginsengnoside profiles of these multiple adventitious roots were similar to profiles of field-grown ginseng roots when analyzed by HPLC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Impact of spatial heterogeneity on a predator-prey system dynamics

This paper deals with the study of a predator-prey model in a patchy environment. Prey individuals moves on two patches, one is a refuge and the second one contains predator individuals. The movements are assumed to be faster than growth and predator-prey interaction processes. Each patch is assumed to be homogeneous. The spatial heterogeneity is obtained by assuming that the demographic parameters (growth rates, predation rates and mortality rates) depend on the patches. On the predation patch, we use a Lotka-Volterra model. Since the movements are faster that the other processes, we may assume that the frequency of prey and predators become constant and we would get a global predator-prey model, which is shown to be a Lotka-Volterra one. However, this simplified model at the population level does not match the dynamics obtained with the complete initial model. We explain this phenomenom and we continue the analysis in order to give a two-dimensional predator-prey model that gives the same dynamics as that provided by the complete initial one. We use this simplified model to study the impact of spatial heterogeneity and movements on the system stability. This analysis shows that there is a globally asymptotically stable equilibrium in the positive quadrant, i.e. the spatial heterogeneity stabilizes the equilibrium.     

Fitness effects of parasite-mediated spatial heterogeneity within a swarm

The fitness consequences associated with the position an individualadopts within a dynamic group are not well understood. I investigatedmate acquisition by male chironomid midges using a simple swarmingmodel and empirically collected data on midge aerobatic ability.Previous work has suggested that the aerobatic ability of amale is an important predictor of his reproductive success,although there is contrary (and counterintuitive) evidence thatinfection with ectoparasitic mites increases a male's chanceof mating, despite having negative effects on flight speed.The model used here suggests that a male's location within theswarm, brought about passively through interindividual differencesin flight speed, may explain these contradictory results. Specifically,slower flying males (including those burdened with mites) adoptedpositions nearer the center of the swarm, whereas faster malestended to occupy the periphery. This in turn affected theiraccess to females because any mechanism that brought femalesnearer the swarm's center before capture (including high femaleflight speed and selective pairing by either males or females)significantly increased the relative reproductive success ofboth larger and parasitized males, with the benefits of parasitismpeaking at around 4 mites per host. There may be selective pressureon hosts and parasites to maintain this optimal mite densitybecause both are likely to benefit from the relationship: hostsenjoy an increased reproductive success, whereas only throughhost copulation can mites transfer to a female midge and returnto water (their next life-history stage) during host oviposition.     

Species coexistence by permanent spatial heterogeneity in a lottery model

We studied the effect of permanent spatial heterogeneity in promoting species coexistence in a lottery model. The system consisted of multiple habitats, each composed of a number of sites occupied by adults of two species. Larvae produced from different habitats were mixed in a common pool. When an adult died, the vacant site became occupied by an individual randomly chosen from the larval pool. If there were n habitats, there could be up to n-1 internal equilibria with both species in addition to two single-species equilibria. These equilibria and their local stability can be calculated from a single function, indicating the difference among species in their average lifetime reproductive success. Our main result is that between-habitat variation in the ratio of mortalities of two species promotes coexistence, while that of reproductive rates does not. This conclusion is the opposite of the role of temporal variation in the standard lottery model, in which between-year variation in the reproductive rate, but not that in the mortalities, promotes coexistence.     

Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns

 In comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we wanted to combine this approach with the promoter-trapping strategy to identify nematode-induced plant promoters. For the efficient production and culture of transgenic hairy root lines of Arabidopsis thaliana, the standard Agrobacterium rhizogenes transformation procedure was modified to avoid rapid callusing of the hairy roots. An average of 0.72 independent kanamycin-resistant (Km^R) roots were obtained per leaf piece. However, a much lower frequency of reporter gene activation was obtained than expected from experiments with the same vectors in Agrobacterium tumefaciens: of more than 700 independent Km^R hairy roots tested, only 8 were β-glucuronidase (GUS) positive. DNA hybridization was done on ten hairy root lines, of which one had a single truncated T-DNA and the others multiple copies of T-DNA that led to complex hybridization patterns. In a parallel analysis of A. thaliana plants transformed with the same vectors using A. tumefaciens, relatively simple T-DNA integration patterns were obtained. The low occurrence of GUS-positive hairy root lines in our experiments could be explained by the multiple T-DNA copies, especially in inverted array, that result in high frequencies of gene inactivation. Received: 11 August 1998 / Revision received: 17 February 1999 / Accepted: 18 March 1999