RESUMEN
Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.
Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Coffea/citología , Equipos Desechables , Glycine max/citología , Nicotiana/citología , Técnicas de Embriogénesis Somática de Plantas/instrumentación , Recuento de Células , Coffea/crecimiento & desarrollo , Diseño de Equipo , Cinética , Oxígeno/metabolismo , Células Vegetales/fisiología , Reología , Glycine max/crecimiento & desarrollo , Nicotiana/crecimiento & desarrolloRESUMEN
Cyperus rotundus L. (Cyperaceae; C. rotundus) is an Indian medicinal plant demonstrated to exert multiple health benefits. The purpose of the present study was to test the biological efficacy of C. rotundus tubers extract on weight control in obese Zucker rats. It was demonstrated that administration of 45 or 220 mg/kg/day of C. rotundus tubers hexane extract for 60 days in Zucker rats induced a significant reduction in weight gain without affecting food consumption or inducing toxicity. In vitro, 250 microg/mL of this extract was able to stimulate lipolysis in 3T3-F442 adipocytes suggesting that this medicinal plant contains activators of beta-adrenoreceptors (AR). The binding assay performed on the rat beta3-AR isoform, known to induce thermogenesis, demonstrated that C. rotundus tubers extract can consistently and effectively bind to this receptor. These data suggest that the effect on weight gain exerted by C. rotundus tubers extract may be mediated, at least partially, through the activation of the beta3-AR. In conclusion, C. rotundus tubers extract prove to be a new herbal supplement for controlling body weight preferentially in beta3-AR sensitive species.
Asunto(s)
Cyperus/química , Obesidad/tratamiento farmacológico , Obesidad/fisiopatología , Tubérculos de la Planta/química , Aumento de Peso/efectos de los fármacos , Adiposidad/efectos de los fármacos , Animales , Biomarcadores , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Lipólisis/efectos de los fármacos , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ratas , Ratas Zucker , Factores de TiempoRESUMEN
The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.
Asunto(s)
Reactores Biológicos , Diseño de Equipo/métodos , Células Vegetales , Reactores Biológicos/economía , Técnicas de Cultivo de Célula , Diseño de Equipo/economíaRESUMEN
A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 microg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Nicotiana/citología , Plantas Modificadas Genéticamente/genética , Virus de la Rabia/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Western Blotting , Técnicas de Cultivo de Célula , Medios de Cultivo/química , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica de las Plantas , Humanos , Proteínas del Tejido Nervioso/aislamiento & purificación , Pruebas de Neutralización , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Nicotiana/virologíaRESUMEN
Soy isoflavones are phytoestrogens which have been associated with several health benefits. In the present study, we report the production of isoflavones in a collection of 40 strains of soya cell cultures established in 1975. A large variability in the isoflavone composition was observed and high-producing strains, with an isoflavone content of up to 46.3 mg g(-1) dry wt., were found. In comparison with soybeans, many callus strains had a higher isoflavone concentration (10-40 times) and a different ratio of genistin to daidzin forms. The highest producing strain was transferred to liquid medium in an Erlenmeyer flask and in a 10 l stirred-tank bioreactor where high isoflavone content (7% dry wt.), concentration (880 mg l(-1)) and a maximum productivity estimated to 60 mg l(-1) d(-1) were obtained. We further studied the estrogenic activity of pure compounds compared to plant cell culture extracts in the estrogen-responsive human endometrial Ishikawa cell line. Estrogen was confirmed to be 1000-10,000 times more active than isoflavones. The estrogenic activity of the extracts correlated to their isoflavone content. The activity of the malonyl isoflavones, assessed here for the first time, was lower than the aglycones. Taken together, these results suggest that soya cell cultures can be used as an alternative source to soybeans to provide high concentrations of bioactive isoflavones.