17 research outputs found
Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures
Background: Liquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase.
Results: In EnBase® cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 μM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase.
Conclusions: PFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation
Perfluorodecalin-supported system enhances taxane production in hairy root cultures of Taxus x media var. Hicksii carrying a taxadiene synthase transgene
Enhanced taxane production was observed in a hybrid, two liquid phases containing, cultures of Taxus x media var. Hicksii hairy root carrying a taxadiene synthase transgene, supported with liquid perfluorodecalin (PFD) in degassed or aerated form. The hairy root cultures were elicited with methyl jasmonate (MJ, 100 μM) or coronatine (COR, 1 μM), and fed with sucrose and l-phenylalanine. The root growth was not stimulated by PFD addition, irrespective of the day of its application (day 0 and 14). However, in the cultures elicited with MJ and performed in the presence of PFD the final root biomass accumulation was higher than in cultures performed without PFD while the opposite effect was observed in cultures supplemented with COR. The highest paclitaxel content in root biomass was determined at the end of the cultures elicited with MJ and supplemented with PFD-degassed at day 0 or 14, 1,440.8 and 1,432.5 μg g−1 DW, respectively. The highest total (i.e. intracellular + extracellular: both in aqueous and PFD phases) paclitaxel yield in flasks (149.15 μg flask−1) was noted after the application of PFD-degassed at day 14. The other taxane detected was baccatin III, only in the root biomass, with the highest content (76.9 μg g−1 DW) observed under COR treatment. Although COR stimulated paclitaxel production with less efficiency than MJ, it resulted in higher paclitaxel excretion to the liquid phases of culture medium and PFD
Does climate affect pollen morphology? : optimal size and shape of pollen grains under various desiccation intensity
Liquid Perfluorodecalin Application for In Situ Extraction and Enhanced Naphthoquinones Production in Arnebia euchroma Cell Suspension Cultures
Morphology and growth of mammalian cells in a liquid/liquid culture system supported with oxygenated perfluorodecalin
Liquid perfluorochemical-supported hybrid cell culture system for proliferation of chondrocytes on fibrous polylactide scaffolds
Modelling of a hybrid culture system with a stationary layer of liquid perfluorochemical applied as oxygen carrier
A mathematical model of a hybrid culture system supported with a stationary layer of liquid perfluorochemical (PFC) as a source of O2 for cells which grow in the aqueous phase of culture medium has been developed and discussed. The two-substrate Monod kinetics without inhibition effects, i.e. the Tsao-Hanson equation, has been assumed to characterise the biomass growth. The Damköhler number which relates the growth rate to the mass transfer effects has been used to appraise the regime (i.e. diffusion-limited or kinetics) of the whole process. The proposed model predicted accurately previously published data on the submerged batch cultures of Nicotiana tabacum BY-2 heterotrophic cells performed in a culture system supported with a stationary layer of hydrophobic perfluorodecalin as a liquid O2 carrier. Estimated values of the parameters of the model showed that the process proceeded in the kinetics regime and the growth kinetics, not the effects of the mass transfer between aqueous phase and liquid PFC, had essential influence on the growth of biomass
Propagation of non-adherent HL-60 cells in batch cultures maintained in staticand wave-type agitated systems
Typically applied static (i.e. non-agitated) cultures do not provide sufficient conditions for efficient propagation of suspended non-adherent cells, in general. Feasibility of small-scale wave-type agitated single-use bioreactors for gentle agitation underlies applicability of such systems for scaling-up of fragile biomass of animal cells. The basic aim of the study was to compare the results of non-adherent HL-60 cell propagation performed referentially as the batch culture in typical static (i.e. non-agitated) disposable culture flasks (50 cm3 of culture medium) and in ReadyToProcess WAVETM25 bioreactor system (GE Healthcare) equipped with disposable culture bag (300 cm3 of culture medium) subjected to continuous wave-type agitation. The density and viability of HL-60 cells were significantly higher for the bioprocess subjected to wave-type agitation, than in the reference static culture. The values of the specific rate of glucose consumption per cell (rglc=cell) exhibited by HL-60 cells maintained in the system with continuous wave-type agitation was significantly lower (i.e. up to more than 42%) than the values noted for the static culture, for exactly the same time-points of two compared cultures. The results of the studies undoubtedly and comprehensively confirmed the applicability of the studied disposable bioreactor with wave-induced agitation as the right platform for proceeding the propagation of non- adherent HL-60 cells and for providing the culture conditions required by HL-60 cells for sustainable metabolism
Oxygen Transfer Effects in a Two-Phase System of an Aqueous Phase and Liquid Perfluorochemical Subjected to Continuous Wave-Assisted Agitation in Disposable Bioreactor
Systems of two immiscible liquid phases—aqueous phase (i.e., distilled water (dH2O) or phosphate-buffered saline (PBS)) and liquid perfluorochemical (i.e., perfluorodecalin (PFD))—were subjected to wave-assisted agitation, i.e., oscillatory rocked, in a disposable bag-like container in a ReadyToProcess WAVETM25 bioreactor, to recognize oxygen transfer effects and effectivity of the surface aeration. According to the DoE methodology, values of the volumetric liquid-side mass transfer (kLa) coefficient for dH2O, PBS, dH2O-PFD, and PBS-PFD systems were determined for the whole range of operating parameters of the WAVE 25 bioreactor. A significantly higher maximal value of kLa was found for waving dH2O than for dH2O-PFD (i.e., 0.00460 s−1 vs. 0.00331 s−1, respectively) compared to more equal maximal values of kLa reached for PBS and PBS-PFD (0.00355 s−1 vs. 0.00341 s−1, respectively). The interface development factor (f) depended on the interfacial area a, and the enhancement factor (EPFD), depending on kLa, was introduced to quantitatively identify the mass transfer effects in the systems of waving two immiscible liquids. The phase of PFD was identified as the reservoir of oxygen. Dimensional correlations were proposed for the prediction of the kLa coefficient, in addition to the f and EPFD factors. The presented correlations, and the set of kLa values, can be directly applied to predict oxygen transfer effects reached under continuous oscillatory rocked systems containing aqueous phase and liquid perfluorochemical