Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa

Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa. Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles. For the heavily aggregated C. calcitrapa, biomass levels [from 10-160 g fresh weight (fw) l(-1))] were linearly correlated with FBRM counts. For M. citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length. For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis. The average aggregate equivalent diameter (C. calcitrapa) and average chain length (M. citrifolia), determined using image analysis, closely tracked the FBRM average chord length. The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.     

Characterization of plant suspension cultures using the focused beam reflectance technique

Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R ²>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.     

Feasibility of Focused Beam Reflectance Measurement (FBRM) for Analysis of Pharmaceutical Suspensions in Preclinical Development

This study examined the use of focused beam reflectance measurement (FBRM) for qualitative and quantitative analysis of pharmaceutical suspensions with particular application to toxicology supply preparations for use in preclinical studies. Aqueous suspensions of ibuprofen were used as prototype formulations. Initial experiments were conducted to examine the effects of operational conditions including FBRM probe angle, probe location, and mixing (method and rate of mixing) on the FBRM analysis. Once experimental conditions were optimized, the homogeneity and sedimentation-redispersion of particles in the suspensions were assessed. Ibuprofen suspension under continuous agitation was monitored using FBRM for 60 h to study particle size change over time. Another study was performed to determine if particle count rates obtained by FBRM could be correlated to suspension concentration. The location and the angle of the FBRM probe relative to the beaker contents, and the rate and the method of mixing the suspension were found to be sensitive parameters during FBRM analysis. FBRM was able to monitor the process of particle sedimentation in the suspension. The attrition of ibuprofen particles was detectable by FBRM during prolonged stirring with an increase in the number of smaller particles and decrease in the number of larger particles. A strong correlation was observed between particle count rate by FBRM and ibuprofen concentration in the suspension. Also, change in content uniformity in the suspension at different locations of the beaker was represented by FBRM particle count. Overall, FBRM has potential to be a useful tool for qualitative and quantitative analysis of pharmaceutical suspensions.     

Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.     

Non-invasive methods for determination of cellular growth inPodophyllum hexandrum suspension cultures

Culture conductivity and on-line NADH fluorescence were used to measure cellular growth in plant cell suspension cultures ofPodophyllum hexandrum. An inverse correlation between dry cell weight and medium conductivity was observed during shake flask cultivation. A linear relationship between dry cell weight and culture NADH fluorescence was obtained during the exponential phase of batch cultivation in a bioreactor under the pH stat (pH 6) conditions. It was observed that conductivity measurement were suitable for biomass characterisation under highly dynamic uncontrolled shake flask cultivation conditions. However, if the acid/alkali feeding is done for pH control the conductivity measurement could not be applied. On the other hand the NADH fluorescence measurement allowed online-in situ biomass monitoring of rather heterogenous plant cell suspension cultures in bioreactor even under the most desirable pH stat conditions.     

The in-line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality.     

The dielectric permittivity at radio frequencies and the bruggeman probe: novel techniques for the on-line determination of biomass concentrations in plant cell cultures

A novel technique is described for the measurement of the volume fraction of biomass in a suspension by the simultaneous measurement of the conductivity of a suspension containing cells and of the medium in which the cells are suspended. The presence of non-conducting particulate matter in a suspension will cause the conductivity of a suspension to be decreased relative to that of the medium in which the particles are suspended. A simple equation (the Bruggeman equation) describes the relationship between the volume fraction of non-conducting particulate matter and the decrease in conductivity. The accuracy of this method for the determination of the biomass concentration of plant cells (Festuca arundinacea) in culture was shown. The method was successfully applied to the on-line determination of biomass concentrations during the growth of F. arundinacea cultures, and gave good agreement with biomass levels as determined from measurements of the radio-frequency dielectric permittivity of such cultures.     

濒危植物珙桐愈伤组织的诱导及悬浮细胞培养初探

以珙桐（Davidia involucrate）无菌苗的下胚轴为外植体,以LSD分析和正交设计与分析为主要研究方法。诱导和筛选愈伤组织,进行悬浮培养,初步建立了珙桐悬浮培养体系,并对珙桐悬浮培养中生物量和pH值的变化进行了初步研究。结果表明：KT和2,4-D的组合诱导出的愈伤组织更适合进行细胞悬浮培养,最佳愈伤组织诱导培养基为WPM＋KT 1.0 mg/L＋2,4-D 0.5 mg/L＋Vc 1.0 mg/L;悬浮细胞培养中,接种量和Ca2＋浓度是影响生物量的主要因素,最佳接种量为25 g/L,最佳Ca2＋浓度为标准WPM培养基中Ca2＋浓度的2倍;黑暗条件下,珙桐悬浮培养生物量变化曲线呈＂S＂型,在21 d时可获得最大生物量;pH值的变化与生物量的变化有一定的相关性。     

不同种类无机盐对灰毡毛忍冬“渝蕾1号”悬浮培养体系中细胞生长和绿原酸含量的影响

为提高灰毡毛忍冬"渝蕾1号"悬浮培养体系中绿原酸的含量,该研究探讨了B_5培养基中不同浓度的无机盐对灰毡毛忍冬"渝蕾1号"悬浮培养细胞生物量及绿原酸含量的影响,通过在悬浮培养体系中添加不同浓度的无机盐,采用重量法测定灰毡毛忍冬"渝蕾1号"悬浮培养细胞的生物量及采用高效液相色谱法测定绿原酸的含量。结果表明:当硝态氮和铵态氮配比与B_5培养基中硝态氮和铵态氮配比一致时,即NO_3~-/NH_4~+摩尔比值为13∶1时,培养体系有利于细胞的生长和绿原酸的积累。当KNO_3浓度为3.5 g·L~(-1)时,细胞生物量达到最大,为19.26 g·L~(-1);当KNO_3在较低浓度(0.5 g·L~(-1)和1.5 g·L~(-1))时,积累较多的绿原酸。NO_3~-的两项研究结果均与对照浓度(2.5g·L~(-1))有一定的差异。另外,对(NH_4)_2SO_4来说,在高于对照浓度0.134 g·L~(-1),即浓度为0.268 g·L~(-1)时,生物量和绿原酸含量都达到了最大。P、Ca、Mg三种矿质元素的研究结果表明,当Na H_2PO_4·2H_2O浓度为0.10 g·L~(-1)、Ca Cl_2的浓度为0.20 g·L~(-1)时,细胞的生长和绿原酸的积累均可达到最大值;而对Mg~(2+)来说,低浓度促进细胞的生长,高浓度促进绿原酸的积累。兼顾细胞生物量和绿原酸含量两个指标,需选择适中的浓度。这些结果均与对照浓度有一定的差异。这说明灰毡毛忍冬"渝蕾1号"悬浮细胞所需无机盐的浓度与B_5培养基无机盐的浓度有一定的差异,选择适宜的浓度可促进其悬浮细胞的生长及次生代谢产物绿原酸的积累。该研究结果为绿原酸的工业化生产打下了基础。     

Rollinia mucosa cell suspension cultures: Establishment and growth conditions

Different types and concentrations of plant growth regulators were tested in order to obtain the best callus and cell suspension culture growth conditions of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Picloram was shown to be the most efficient for induction and production of friable calluses, independent of the concentration used. Cellular morphology and viability, fresh and dry weights, pH and medium sugar concentration were determined for cell suspension cultures. Dissimilation curves were used for the characterization of the growth of cell suspension cultures. Picloram provided the most rapid growth and produced the highest biomass, with little variation in morphology (differentiated cells). It also provided the highest dissimilation, when compared with cell suspension cultures maintained in media with 2,4-D or NAA + BA + GA₃. Stable cell suspension cultures can be established in MS medium supplemented with 20.8 μM picloram. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of plant-based expression platforms for the heterologous production of geraniol

We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 μg/g fresh weight, fw), followed by the transient expression system (27 μg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 μg/g fw), and finally hairy root cultures (9 μg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 μg/g fresh biomass per day, whereas transient expression produced 5.9 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol.     

Growth behavior in plant cell cultures based on emissions detected by a multisensor array

The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.     

On-line biomass monitoring by capacitance measurement.

An in-situ, steam-sterilizable capacitance probe was used to follow the biomass concentration on-line, in bioreactors from 20 to 2000 l total volume. Microbial cultures of Saccharomyces cerevisiae, Pichia pastoris and Streptomyces virginiae were grown in batch and fed-batch culture in both defined and complex media in order to demonstrate the wide dynamic operating range of the instrument. A linear correlation was found between the on-line capacitance measurement and the off-line measurements (optical density, OD620; packed mycelial volume, PMV; biomass concentration X, and colony forming units, CFU ml-1) for biomass concentrations (dry cell weight) up to 30 g l-1 (St. virginiae), 106 g l-1 (S. cerevisiae) and 89 g l-1 (P. pastoris). The on-line capacitance measurement was slightly influenced by variations in agitation speed and strong extraneous radio frequencies. A specific capacitance constant (Cs) was defined for all microbial cells which was dependent on cell viability and cell size. The Cs was easy to calculate using the on-line capacitance measurement and an off-line estimation of biomass concentration. The Biomass Monitor proved suitable for precise on-line monitoring of both homogeneous (uni-cellular) and heterogeneous (mycelial) cultures in bioreactors.     

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.     

Influence of endophytic fungal elicitation on production of inophyllum in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

The influence of dried cell powder and culture filtrates of endophytic fungi on production of inophyllum in cell suspension cultures of leaf- and stem-derived callus of Calophyllum inophyllum was investigated. Two fungi, Nigrospora sphaerica and Phoma spp., endophytic to C. inophyllum, were isolated from leaf tissues, and were identified by both 18S rRNA gene amplification and sequencing. Elicitation of suspension cultures of both callus types of C. inophyllum with dried cell powder and culture filtrates of both fungi consistently elicited production of inophyllum A, B, C, and P. In comparison to stem-derived callus, suspension cultures of leaf-derived callus enhanced production of most inophyllum. Of the four inophyllum studied, the highest production of inophyllum A, C, and P was achieved in elicited suspension cultures of leaf-derived callus. Suspension cultures of stem-derived callus enhanced production only of inophyllum B. When suspension cultures of leaf-derived callus were elicited with 40 mg dried cell powder of Phoma spp., a level of 751-fold (6.84 mg/100 g elicited biomass) of inophyllum A was produced, compared to control. Whereas, a level of 414-fold (6.22 mg/100 g elicited biomass) of inophyllum B was produced when suspension cultures of stem-derived callus were elicited with 20 mg dried cell powder of N. sphaerica. When compared to control, a 10% culture filtrate of N. sphaerica in suspension cultures of leaf-derived callus elicited inophyllum C and P production by 928-fold (7.43 mg/100 g elicited biomass) and 750-fold (1.5 mg/100 g elicited biomass), respectively.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

Production of L-DOPA by cell suspension cultures of Mucuna pruriens

The endogenous synthesis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) by cell suspension cultures of Mucuna pruriens was found to be influenced by several environmental parameters. The nature of the nitrogen source as well as the concentration of nitrogen containing salts, sucrose and phosphate in the culture medium were found to affect the biosynthesis of L-DOPA. Addition of 2, 4-dichlorophenoxyacetic acid to the medium suppressed L-DOPA production; continuous illumination of the cultures had a strong beneficial effect on L-DOPA production. L-DOPA was accumulated intracellularly by the cell suspension cultures. These observations further demonstrate that for certain products of plant cell suspensions product synthesis can be manipulated by a proper selection of specified nutrients.     

细胞均一性对葡萄细胞生长和花青素合成的影响

通过色差筛选法建立了一个相对均一的葡萄细胞悬浮系E,其细胞团较小,在长期继代培养过程中花青素合成能力的变异系数为8.7%,重复摇瓶实验的变异系数为5%。以E为实验材料进行的各组前体饲喂、诱导子添加、光照等联合作用实验,其生物量和花青素合成的变异系数均可控制在12%以内,充分说明了培养体系的均一性对维持稳定生产的重要性;黑暗条件下添加30μmol/L苯丙氨酸(Phe)和218μmol/L茉莉酸甲酯(MeJA)可使单位细胞花青素含量达到对照组的5.89倍,花青素产量为对照组的4.30倍,且连续5次继代培养过程中生物量和花青素合成的变异系数均比对照组降低。     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.