Real-time compensation of the inner filter effect in high-density bioluminescent cultures

Bioluminescence has recently become a popular research tool in several fields, including medicine, pharmacology, biochemistry, bioprocessing, and environmental engineering. Beginning with purely qualitative goals, scientists are now targeting more demanding applications where accurate, quantitative interpretation of bioluminescence is necessary. Using the recent advances in fiber-optic technology, bioluminescence is easily monitored in vivo and in real time. However, the convenience of this measurement is often concealing an unsuspected problem: the bioluminescence signal might be corrupted by a large error caused by the extinction of light by biological cells. Since bioluminescent cultures not only emit light but also absorb and scatter it, the measured signal is related in a complex, nonlinear, and cell-concentration-dependent manner to the "true" bioluminescence. This light extinction effect, known as the "inner filter effect," is significant in high-density cultures. Adequate interpretation of the bioluminescence signal can be difficult without its correction. Here, we propose a real-time algorithm for elimination of the inner filter effect in a bioreactor. The algorithm yields the bioluminescence which would be measured if the glowing culture was completely transparent. This technique has been successfully applied to batch and continuous cultivation of recombinant bioluminescent Escherichia coli. (c) 1993 John Wiley & Sons, Inc.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Correcting for the inner filter effect in measurements of fluorescent proteins in high-cell-density cultures

Fluorescent proteins (FPs), such as green fluorescent protein (GFP) and its variants, are well-developed visible markers for analyzing bioprocesses. Accurate measurement of fluorescence emitted from FPs in whole cells is complicated by the inner filter effect (IFE), which is caused by intracellular light absorption and scattering by cell particles. The IFE causes nonlinearity between fluorescence intensity and fluorophore concentrations in FP-harboring cells and can significantly influence the accuracy of FP-based analysis, especially at high cell densities. A mathematical model based on detection of fluorescence intensity using a fluorescence spectrophotometer was developed to provide a simple correction for the IFE in fluorescence intensity detection in high-density cultures. The parameters of this model were determined in three different FP-harboring bacterial strains to give the “real fluorescence” intensity without the IFE. Using these parameters, accurate analysis of FP-labeled Escherichia coli at high cell density in pure culture and in mixed cultures with fluorescent and nonfluorescent strains was easily and successfully achieved.     

High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.     

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins.     

Simple and sensitive fluorescence sensing of methyl parathion based on the inner filter effect of p-nitrophenol on nitrogen-doped titanium carbide quantum dots

It is of great significance to develop an effective method for methyl parathion (MP) detection. Herein, a novel nitrogen-doped titanium carbide quantum dots (N-Ti₃C₂ QDs) was prepared and used to construct a simple and sensitive fluorescence sensing platform of MP by making use of inner filter effect (IFE). The prepared N-Ti₃C₂ QDs can exhibit strong blue fluorescence at 434 nm. Meanwhile, MP could hydrolyze to produce p-nitrophenol (p-NP) under alkaline conditions, which showed a characteristic ultraviolet-visible (UV-visible) absorption peak at 405 nm, resulting in the fluorescence of N-Ti₃C₂ QDs is effectively quenched by p-NP. In addition, the investigation of time-resolved fluorescence decays indicated that the corresponding quenching mechanism of p-NP on N-Ti₃C₂ QDs is due to the IFE. After optimizing the conditions, the as-developed fluorescence sensing platform displayed wide detection range (0.1–30 μg mL⁻¹) and low detection limit (0.036 μg mL⁻¹) for MP, and it was also successfully applied for MP analysis in real water samples, thus it is expected that this simple, sensitive and enzyme-free sensing platform shows great applications.     

Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect

Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it loses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, K(m), and k(cat). The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate's extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive k(cat) and K(m) from progress curves. Strategies to obtain reliable and reproducible estimates of k(cat) and K(m) from only two or three progress curves are illustrated using matrix metalloproteinase 12 and alkaline phosphatase. Accurate estimates of concentration of enzyme-active sites and specificity constant k(cat)/K(m) (from one progress curve with [S]相似文献   

气体成分对植物细胞悬浮培养的影响

气体成分对植物悬浮培养细胞的生长和次生代谢物的产量有深刻的影响。就有关氧、二氧化碳、乙烯和一些未知成分作用的研究进行了综述。     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.     

Production of recombinant h-AT III with mammalian cell cultures using capillary electrophoresis for product monitoring

The importance of mammalian cell cultures for biotechnological production processes is steadily increasing, despite the high demands of these organisms on their culture conditions. Efforts towards a more efficient bioprocess generally concentrate on maximizing the culture's life time, the cell number, and the product concentration. Here recombinant BHK 21 c13 cells are used to produce rh-AT III, an anticoagulant of high therapeutic value. The influence of the process mode (batch, repeated batch, continuous perfusion) and the process temperature (30°C vs. 37°C) on the above mentioned parameters is investigated. It is possible to increase the length of the culture from 140 h (batch) to more than 500 h (continuous perfusion culture), while concomitantly increasing the cell density from 0.72 10⁶/ml (batch) to 2.27 10⁶/ml (repeated batch) and 2.87 10⁶/ml (continuous perfusion culture). The accumulation of toxic metabolites, such as lactate, can be curtailed by reducing the bioreactor temperature from 37°C to 30°C during the later part of the exponential growth phase. Fast and reliable product monitoring became essential during process optimization. Capillary zone electrophoresis (CZE) in uncoated fused silica capillaries was studied for that purpose and compared to the standard ELISA. Under optimized conditions an AT III quantification could be done within 2 min with CZE. The detection limit was 5 g/ml. A relative standard deviation of less than 0.9% was calculated. The detection limit could be lowered by one order of magnitude by using a two dimensional system, where an liquid chromatographic (LC) system is coupled to the CZE. Concomitantly the resolution is improved. The two-dimensional analysis required 5 min. Membrane adsorbers (MA) were used as stationary phase in the LC-system, to allow the application of high flow rates (5–10 ml/min). The correlation between the LC-CZE analysis and the standard AT III-ELISA was excellent, with r²: 0.965. Using the assay for at line product monitoring, it is shown, that the process temperature is of no consequence for the productivity whereas the process mode strongly influences this parameter.     

刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用

刺激剂（ｅｌｉｃｉｔｏｒ）在植物细胞培养中被用来作为提高次生代谢物产量的手段。文中概括介绍了微生物、寡聚糖、蛋白质、第二信使及其他物质作为刺激剂在植物细胞培养中的应用及其研究成果。     

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andK _hGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andK _hGM-CSF of 7.64 after 2 h of incubation at room temperature.     

Monitoring and control of the physiological state of cell cultures

Advances in bioprocess engineering depends ultimately on the level of understanding and control of the physiological state of the cell population. Process efficiency is strongly influenced by changes in the cellular state which should be monitored, interpreted, and, if possible, properly manipulated. In most control systems this function is not explicitly considered, which hampers process development and optimization. Conventional control logic is based on direct mapping of the growth environment into process efficiency, thereby bypassing the cell state as an intermediate control objective. Today, this limitation is well realized, and explicit monitoring and control of cellular physiology are considered to be among the most challenging tasks of modern bioprocess engineering. We present here a generic methodology for the design of systems capable of performing these advanced monitoring and control functions.The term "physiological state" is quantified by a vector composed of several process variables that convey significant information about cellular state. These variables can be selected among different classes, including specific metabolic rates, metabolic rate ratios, degees of limitation, and others. The real-time monitoring of many of these is possible using commercial sensors. The definition and calculation of representative sets of physiological state variables is demonstrated with examples from several fermentor cultures: recombinant Escherichia coli for phenylalanine production, bioluminescent E. coli (harboring lux genes driven by a heat shock protein promoter) for detection of environmental pollutants, plant cell culture of Perilla frutescensfor anthocyanin production, and perfusion cultures of recombinant mammalian cells (NS0 and CHO) for therapeutic protein production.If the physiological state vector is on-line calculated, the fermentation process can be described by its trajectory in a space defined by the vector components. Then, the goal of the control system is to maintain the physiological state of the cell as close as possible to the trajectory, providing maximum efficiency. A control structure meant to perform this function is proposed, along with the mechanism for its design. In contrast to conventional systems which work in a closed loop in respect to the cell environment, this scheme operates in a closed loop in respect to the cell state. The discussed control concept has been successfully applied to the recombinant phenylalanine production, resulting in physiologically consistent operation, total computer control, and high process efficiency. Initial results from the application of the method to perfusion mammalian cell cultures are also presented. (c) 1996 John Wiley & Sons, Inc.     

Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams

Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc.     

An improved polyurethane support system for monitoring growth in plant cell cultures

An improved culture system for plant cells that employs filter paper resting on polyurethane saturated with liquid medium is described. It combines a simplified version of the system outlined by Weber and Lark [1979, Theor Appl Genet 55: 81–86] with the method of growth estimation described by Horsch et al. [1980, Can J Bot 58: 2402–2406]. The growth of plated cells or callus can be conveniently monitored through repeated non-destructive fresh weight measurements of the filter paper and adhering cells, thereby allowing the construction of a complete growth curve over the course of an experiment. Experiments with 3 Nicotiana genotypes (N. plumbaginifolia Viv., N. tabacum L. SC 58 and N. tabacum WI 38) and 3 Vitis vinifera L. genotypes (Chenin Blanc, Dogridge and White Riesling) clearly demonstrate higher growth rates of plated cells on polyurethane supports compared with agar. Further experiments with N. plumbaginifolia illustrate the use of polyurethane supports for culturing cells at low pH (4.0) and the recovery of spent medium for monitoring changes in pH. These features will greatly facilitate quantitative studies of mineral nutrition and metal toxicity in cultured cells. Polyurethane supports also allow the incorporation of conditioned medium or feeder cells to support the growth of cells at low densities and facilitate the rapid recovery of variant cells.     

Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis

Fractional precipitation is a simple and efficient procedure for pre-purification of paclitaxel. The optimal methanol composition in water, paclitaxel content in crude extract, storage temperature, and time were 61.5% (v/v), 0.5% (w/v), 0°C, and 3 days, respectively. As purity of the paclitaxel in the crude extract increases, the purity and yield of paclitaxel from fractional precipitation increases. This pre-purification process serves to minimize the solvent usage, size and complexity of the HPLC operations for paclitaxel purification. This process is readily scalable to a pilot plant and eventually to a production environment where multikilogram quantities of material are expected to be produced.     

Reduction of medium consumption in perfusion mammalian cell cultures using a perfusion rate equivalent concentrated nutrient feed

Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 10⁶ cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L⁻¹ day⁻¹ during a short stable phase, and versus 0.46 g L⁻¹ day⁻¹ in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing set-up to enable better control on nutrient addition.     

Recent advances in plant cell cultures in bioreactors

Two key issues in the application of plant-cell-culture technology to the production of valuable secondary metabolites are reviewed: the selection of cell lines with suitable genetic, biochemical and physiological characteristics; and the optimization of bioreactor environments. Although great progress has been made in recent years in the design, selection and optimization of bioreactor hardware, optimization of environmental factors such as medium components, light irradiation and O₂ supply needs detailed investigations for each case. With a better understanding of plant cell metabolism and physiology, further developments in cultivation processes, such as process integration and on-line monitoring and control, can be expected in the near future.J.-J. Zhong and J.-T. Yu are with the Research Institute of Biochemical Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China T. Yoshida is with the International Center of Cooperative Research in Biotechnology (ICBiotech), Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

Intrinsic glycosylation potentials of insect cell cultures and insect larvae

Summary The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. TheSpodoptera frugiperda (Sf21) andTrichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. Similarly, theT. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides. Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus inT. ni larvae was modified with complex oligosaccharide having sialic acid linked α(2–6) to galactose.