Viability characterization of Taxus chinensis plant cell suspension cultures by rapid colorimetric- and image analysis-based techniques
For the commercially established process of paclitaxel production with Taxus chinensis plant cell culture, the size of plant cell aggregates and phenotypic changes in coloration during cultivation have long been acknowledged as intangible parameters. So far, the variability of aggregates and coloration of cells are challenging parameters for any viability assay. The aim of this study was to investigate simple and non-toxic methods for viability determination of Taxus cultures in order to provide a practicable, rapid, robust and reproducible way to sample large amounts of material. A further goal was to examine whether Taxus aggregate cell coloration is related to general cell viability and might be exploited by microscopy and image analysis to gain easy access to general cell viability. The Alamar Blue assay was found to be exceptionally eligible for viability estimation. Moreover, aggregate coloration, as a morphologic attribute, was quantified by image analysis and found to be a good and traceable indicator of T. chinensis viability.
KeywordsTaxus chinensis plant cell culture Plant cell aggregates Viability Aggregate coloration Alamar Blue assay Image analysis
Commercial systems for cultivation of plant cells were established more than 25 years ago. Significant advancements have been made in understanding metabolite production in large-scale plant cell cultures, but so far controlling variability in product accumulation has often been neglected in favor of improving yield . For the industrially established process of paclitaxel production by submerse cultivation of Taxus chinensis plant cells, a reliable and applicable way to measure the important process parameter of the plant aggregate size with <100 μm up to over 2 mm was established and evaluated by laser diffraction technique ; an alternative way to measure aggregate size had previously been established by Kolewe et al. . The morphology of these aggregates was of some importance, because aggregate size has been proposed to correlate with productivity [4, 5], whereas aggregate coloration was reported to depend on cell age and viability . However, no clear connection between aggregate coloration and viability has been shown so far.
For processes with T. chinensis plant cells, no viability estimating techniques fit for industrial application have so far been established, besides measurements of cell fresh weights and cell dry weights. A reduction of cell growth, however, does not imply a reduction of viability  and zero growth is not necessarily an indicator of poor viability. Since plant cells in suspension mainly stay connected after division and grow in aggregates, the determination of the number of cells is virtually impossible. Viability assays are based either on the physical properties or biochemical characteristics of the cells. The detection methods are based on the properties of the cells, such as endocytosis, enzymatic activity, the integrity of the cell membrane, or proliferation. There are generally different ways to measure the viability of cells. It is possible to obtain information on cell viability by observation of the cytoplasmic flow; membrane integrity can be determined by electrolyte flow, plasmolysis and dye exclusion or retention . Furthermore, the biochemical activity can be measured as in protein synthesis, tetrazolium chloride reduction, DNA and RNA synthesis and fluorescein-di-acetate staining . Microscopic measurements, based on the color of cells incubated with Evans Blue or Trypan Blue, are possible. Dead cells have a disrupted membrane, and can therefore take up the dye . Currently, such tests are popular for mammalian cell culture, as they allow easy automation.
A major breakthrough in the field of biochemistry-based viability tests was achieved by the development of assays based on the activity of the electron transport chain . Such assays based on water-soluble, colorless tetrazolium salts such as 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) and 2.3.5-triphenyltetrazolium chloride (TTC) are popular for mammalian and plant cell cultures. When entering a living cell, TTC is reduced by the dehydrogenase activity of the mitochondrial electron transport chain to form insoluble red formazan product. Therefore, dead and living cells can be distinguished by color. For quantification, the red precipitate is extracted from the tissue with ethanol, and the absorbance of the extract can be determined spectrophotometrically . The TTC has already been shown to be suited for the viability determination of Taxus cultures [12, 13]. Like the TTC test, the MTT assay is based on the dehydrogenase activity of the mitochondrial electron transport chain. Here, the yellow-colored tetrazole of MTT is converted to water-insoluble purple formazan. To dissolve the water-insoluble product, organic solvents such as DMSO or isopropanol are required. The intensity of the blue color can be directly correlated to the number of living cells . All tetrazolium salts are generally toxic because the formazan crystals that are produced from reduction of the salts must be solubilized with DMSO or HCl/isopropanol which lead to cell death [15, 16]. However, in addition to the toxic character of the applied chemical viability assays like the MTT-based one require multiple steps. Therefore, a more simple and non-toxic method would be favorable for standard applications in terms of monitoring industrial processes as well as for process development.
Recently, the Alamar Blue (AB) dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity . AB is a non-toxic redox indicator, which can be detected by both fluorescence and by color change. It contains resazurin and resorufin as oxidation–reduction indicators. The oxidized blue, non-fluorescent form of resazurin is converted by reduction into a pink fluorescent dye resorufin . The reduction of AB occurs intracellularly involving reductases and the mitochondrial electron transport chain . AB acts as an electron acceptor between cytochrome a,a3 and the final reduction step of O2, and does not interfere with the function of the respiratory chain . Other tetrazolium salt-based agents like MTT and TTC have a more negative redox potential than the components of the electron transport chain, and thus consume energy in form of ATP during the reduction. AB on the other hand possesses a more positive redox potential than FMNH2, FADH2, NADH, NADPH and the cytochromes. Thus, a reduction is possible without additional energy requirement . The ‘resazurin reduction test’ has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality . The AB assay has so far been used for the determination of the viability of bacteria, mycobacteria, fungi and several human cell lines [18, 19, 20, 21, 22]. It was also used to assess the viability of tomato plant cells .
There has been an ongoing speculation, whether specific aggregate coloration might be related to cell viability, because such a link might be easily exploited for easy and rapid viability estimation. Specific aggregate colorations have previously been used for characterization of calli, as they are often an indicator on the specific potential of the callus . Callus color may change under varying medium composition . Thus, the color of suspended cells varies, which can be used for evaluation purposes . Especially in suspension cultures for pigment production the color of cells is directly related to their quality . Taxus suspension cultures have furthermore been reported to change appearance in color after exposure to light . Paclitaxel production in calli depends on morphology and age . The callus color was also previously found to correlate with variability in cell growth. Light brown calli showed a higher growth rate than dark and aggregated calli . However, calli were found to produce more paclitaxel at an old age with brown coloration, than at a younger age without the brown stain [28, 29, 30]. The intensification of darkening at the stationary growth phase may be related to increased biosynthesis of phenolic compounds .
The aim of the present study was to elucidate the relationship between T. chinensis aggregate coloration and cell viability. To this end the AB assay had to be established as a non-toxic, rapid, robust and reproducible viability test for cultures of T. chinensis, because no viability assays for industrial application had been previously established. Consequently viability data gained from aggregate coloration through microscopy and automatic image analysis were compared and verified by the AB assay.
Materials and methods
Conditions for cultivation of Taxus chinensis
Gamborg B-5 Basal Medium  was used for cultivation of T. chinensis plant cells described by Wucherpfennig et al.  in 1L polycarbonate Erlenmeyer flasks with vent caps (Corning, USA), filled with 400 mL medium. Cultures were incubated for 240 h at 120 min−1 on a rotary shaker (Certomat BS-1/50 mm, Sartorius, Göttingen, Germany). Cultivations were carried out in triplicate. The growth temperature was 25 ± 0.1 °C.
Alamar Blue assay for T. chinensis viability determination
Since the AB assay has so far not been used for T. chinensis plant cells, it was necessary to adjust the assay for analysis of Taxus cell aggregates. The incubation time, the age of the culture used for generating the calibration curve, lighting conditions and type of incubation were identified to significantly influence measurement results and reproducibility of the test. These factors were gradually optimized. Byth et al.  found that common plant cell culture media and buffers reduce the AB reagent (CellTiter-Blue Cell Viability Assay Kit, Promega, Madison, USA). Therefore, it was necessary to filter cells (Miracloth filter, pore size 25 μm, Calbiochem, Germany) and rinse them in 10 mL deionized water; 50 mg of Taxus cells was suspended in 200 μL, pH 7.2 phosphate buffer. AB reagent was used in a final concentration of 10 % (v/v) within the suspension. After incubation (see "Results and discussion") the cells were disrupted for 60 s in an ultrasonic bath and centrifuged at 12,000 min−1 for 10 min (Biofuge pico, Heraeus, Thermo Fisher Scientific, Waltham, USA) at room temperature. Before fluorescence measurement done at room temperature as well, the samples were shaken in the fluorescence reader (Fluoroskan Ascent, Thermo Scientific) for 10 s at 180 min−1 and measured at 544 nm excitation and 590 nm emission wavelength, respectively. For evaluation of optimal incubation of the assay time resorufin absorbance was measured at 540 and 620 nm (SPECORD® 200, Analytik Jena AG, Jena, Germany). Since the oxidized and reduced spectra are overlapping the equation introduced in Byth et al.  was used for calculation of the reduction value (%).
To compare different methods of controlled eradication of plant cells, 50 mL of cell suspension was taken from a shaken culture. The cells were filtered through a water jet pump with a suction filter and the filter cake was subsequently rinsed with 10 mL distilled water; 250 mg wet biomass was each weighed and given into 1.5 mL tubes. For the eradication experiments by UV light, freezing and heating to 40 °C respectively, 900 μL of phosphate buffer was introduced into the reaction vessels. For eradication by ethanol, 450 μL phosphate buffer and 450 μL of 70 % ethanol were used. To exterminate cells using a saline solution, 250 mg wet biomass was mixed with 900 μL, 5 % NaCl (w/w) solution. All samples were incubated for 1 h at room temperature.
To determine the validity of the AB assay for T. chinensis suspension cultures, a comparison with the MTT assay, already established for plant cells , was conducted. For the preparation of the MTT reagent, 5 mg thiazolyl blue tetrazolium bromide powder (Sigma-Aldrich, St. Louis, USA) was dissolved in 1 mL deionized water. For performing the MTT assay, the cell culture suspension was filtered, rinsed and re-suspended in buffer as described for the AB assay. Each 100 μL of this suspension was transferred to a 96-well plate using a wide-nozzle pipette. After the addition of 10 μL of MTT reagent to each well the plates were incubated in a photometer (Sunrise microplate reader, Tecan, Maennedorf, Switzerland) at 37 °C for 2 h in the dark. After incubation, 100 μL of a DMSO/acidic isopropanol mixture 1:1 (v/v) was added to the wells. The samples were further incubated at 37 °C and measured every 30 min at 450 nm.
Microscopy and image analysis of T. chinensis samples
Mechanical stress experiments
Results and discussion
Development of a dye-based method for assessment of Taxus chinensis plant cell viability
The AB assay is a one-step, extremely simple, reproducible, economical, and non-toxic procedure to evaluate cell proliferation and survival . The AB assay offers various advantages in comparison to other more established assays, including technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity . Further advantages of the AB assay include the water solubility of the formazan product, economic efficiency and minimal toxicity . To establish the AB assay for T. chinensis cultures, a calibration graph with a known composition of viable and dead cells and corresponding fluorescences had to be established. To this end, Taxus cells were eradicated in a controlled manner by incubation for 1 min in liquid nitrogen. Subsequently cells were gently defrosted in warm (37 °C) water and then blended with living cells to create the desired ratios of living cells. The proportions of living cells were normalized and arranged between 0 (all dead) and 1 (highest fluorescence measured, presumably all living).
To further improve the reproducibility of the assay, the samples were mixed during the entire incubation period in an overhead rotator (Revolver Tube Mixer, Labnet International, Inc. Edison, USA), to make sure that aggregates were suspended at all times. The rotating incubation was a reason for the generally high reproducibility of the assay, with deviations of less than 1 % between three measurements.
Viability of T. chinensis plant cell suspension cultures using image analysis technique
Comparison of dye- and image analysis-based viability methods
The hypothesis that the viability of the plant cell culture correlated with the browning of the cells could be confirmed by the comparison of the image analysis and the AB assay. In general, both methods show similar results and are comparable. In comparison to the biochemical assay, the image analysis technique underestimates viability somewhat (Fig. 7b), variation of absolute values is also dependent on standardization. The presence of UV-light or NaCl did not lead to a significant reduction in viability, as the percentage of living cells measured with the AB assay remained at around 90 % and approximately at 80 % measured by image analysis. The addition of 70 % ethanol in a ratio of 1:1 (v/v) resulted in a significant reduction in cell viability to around 60 %. An increase in temperature to 40 °C led to a decrease in viability to around 80 % measured by the AB assay, and about 60 % as measured by image analysis. Freezing led to an eradication of all cells, following the AB assay and produced a rest viability of 10 %, measured by image analysis.
Production of metabolites via plant cell suspension culture is a renewable, environmentally friendly, and economically feasible alternative for extraction from whole plant material . Advancements have been made in understanding metabolite production in large-scale plant cell cultures, but so far there is still a high optimization potential to increase final product titer. The introduced AB assay was found to be exceptionally eligible for viability estimation in industrial processes. Moreover, aggregate coloration, as a brightness attribute, was identified as a good indicator of viability using image analysis methods. Both methods provide useful techniques for determining the viability at different physico-chemical perturbations on T. chinensis cell aggregates.
By optimizing the process parameters incubation time, place, type and age of the cell culture used for generating the calibration curve, higher fluorescence values and more reproducible results for the AB assay could be achieved. By image analysis, the percentage of dead cells could be reliably measured over a grayscale balance between the living and dead cell areas. Both methods of viability determination were compared and led to similar conclusions. However, compared to the AB assay the image analysis technique exhibited higher error values while discrepancies between differently treated samples were less significant.
In total, both AB assay and image analysis offer significant advantages to already established viability assays in plant cells. The AB assay is not destructive or generates toxic samples. It offers many advantages over tetrazolium salts and omits their incompatibility issues . It also has a higher sensitivity at lower percentage viability. The flexibility in the quantification method—fluoro- or spectrophotometrically—is also advantageous . It could be observed in the experiments carried out, that the deviations of triplicates of the AB assay were significantly lower than the error values in the MTT test.
For application, the two viability assays were successfully introduced to determine the influence of different physico-chemical perturbations, such as UV, chemical additives, salt load, high and low temperature and stirrer-induced shear stress, on the viability of T. chinensis suspension cultures. The AB assay was found to be exceptional eligible for viability estimation in industrial processes. Moreover, aggregate coloration, as a brightness attribute, was identified to be clearly related to T. chinensis plant cell viability. Image analysis of culture broth photographs evaluating the brownish color of the aggregates provides an easy and fast way for viability estimation especially well suited for industrial application.
The setup used in this study provided a good way to quantify the influence of hydrodynamic stress within a stirred tank bioreactor on plant cell culture viability. Generally, hydrodynamic stresses lead to a decline not only in viability, but in aggregate size as well. An application to measure aggregate size of plant cells using the technique of laser diffraction was recently introduced by Wucherpfennig et al. . This system could under non-growth conditions (suspension of aggregates in sodium chloride solution) also be used to study various stirrers and reactor geometries for their induced shear stress and effect on plant aggregate integrity. A similar approach was taken by Eslahpazir et al.  using pellets of A. niger. Usually the disintegration effect of mechanical stress and power input is measured using a shear sensitive clay polymer floc system [49, 50]. However, the employment of real biological aggregates has obvious advantages like analogous particle rigidity, size and form. Thus, comminution of native Taxus aggregates might provide further insight about shear susceptibility of biological samples and might complement and expand the results of this study.
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