Population and biomass kinetics in fed-batch cultures of Daucus carota L. somatic embryos

The population dynamics of developing somatic embryos of carrot (Daucur carota L.) was investigated in batch and fed-batch cultures using modified Murashige and Skoog medium. These substrate limitations coincided not only with stoppage of biomass increase, but also with the increase in total concentration of embryos as well as the advancement of the embryo into a more mature stage. Both glucose and ammonium were depleted from the culture. Restoring either glucose, or ammonium and nitrate, as to approximately initial concentrations in fed-batch experiments, did not result in a significant increase of the total normal embryo concentration. On the other hand, medium replacement led to increase in biomass concentration, total embryo number, and improved embryo maturity. The addition of a mixture of glucose, ammonium, and nitrate to the spent medium resulted in variable increases in biomass and embryo number, but always less than those resulting from media replacement. Although the total number of embryos was higher after medium replacement, the fraction of embryos reaching torpedo stage was still only 50%. The need for a better means of population characterization for further kinetic studies is discussed. (c) 1993 Wiley & Sons, Inc.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Influence of boron deficiency on 3H indole-3yl-acetic acid uptake and efflux in cell cultures of Daucus carota L.

³H-IAA net uptake and efflux was measured in carrot cell suspensions after two and five days in either boron (B) sufficient or B deficient media. Contrary to earlier observations with substances such as phosphate, glucose and potassium (or Rb⁺), ³H-label uptake from IAA over a 3 h period did not differ significantly after two days of treatment. After five days of B deficiency, cells accumulated higher ³H-activity than those receiving B. Total ³H-efflux from deficient and sufficient cells did not differ significantly but B deficient cells released more water soluble (pH 3.0) ³H, and less methanol soluble ³H activity, which may be due to a higher IAA oxidase activity. Correspondingly, we found an elevated peroxidase (POD) activity in B deficient cell cultures which was 54% higher after two days of deficieny and which increased further threefold after five days. Similar changes have been found in the amount of POD activity released to the nutrient solution with even more pronounced differences after two days. Thus, B deficient cells released less authentic ³H-IAA (1.7% vs. 3.1% related to total ³H efflux), as determined by HPLC separation.     

Ensuring oxygenation of carrot cell cultures in a Couette viscometer during investigation of shear effects

Mathematical simulation and experimental measurement of dissolved O₂ were performed for extended (up to 8 h) shear testing of Daucus carota (carrot) cell cultures in a conventional Couette viscometer (0.625 mm annulus). The results suggest O₂ depletion below critical levels for cell growth may occur. A novel design modification incorporating an O₂-permeable silicone-layer spun cast on a porous ceramic bowl was devised. It significantly improved oxygenation of the cell cultures, keeping dissolved O₂ near saturation.     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors

The short-time effects of shear on suspended cells of Perilla frutescens were quantitatively analyzed by exposing the cells to a well-defined flow field in a rotating drum reactor. It was found that both shear rate and shearing time significantly affected cell viability. The quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the cell cultures were further investigated in a series of batch cultivations using a 5-L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of 20 to 30 s(-1) or an impeller tip speed of 5 to 8 dm/s, which maximized all the values of the following parameters: the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. (c) 1994 John Wiley & Sons, Inc.     

Oxidation and nuclear localization of thioredoxin-1 in sparse cell cultures

Reactive oxygen species (ROS) were once viewed only as mediators of toxicity, but it is now recognized that they also contribute to redox signaling through oxidation of specific cysteine thiols on regulatory proteins. Cells in sparse cultures have increased ROS relative to confluent cultures, but it is not known whether protein redox states are affected under these conditions. The purpose of the present study was to determine whether culture conditions affect the redox state of thioredoxin-1 (Trx1), the protein responsible for reducing most oxidized proteins in the cytoplasm and nucleus. The results showed that Trx1 was more oxidized in sparse HeLa cell cultures than in confluent cells. The glutathione pool was also more oxidized, demonstrating that both of the major cellular redox regulating systems were affected by culture density. In addition, the total amount of Trx1 protein was lower and the subcellular distribution of Trx1 was different in sparse cells. Trx1 in sparse cultures was predominantly nuclear whereas it was predominantly cytoplasmic in confluent cultures. This localization pattern was not unique to HeLa cells as it was also observed in A549, Cos-1 and HEK293 cells. These findings demonstrate that Trx1 is subject to changes in expression, redox state and subcellular localization with changing culture density, indicating that the redox environments of the cytoplasm and the nucleus are distinct and have different requirements under different culture conditions.     

Metabolic flux analysis of CHO cell metabolism in the late non‐growth phase

Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non‐growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non‐growth phase was performed by a combination of steady‐state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non‐growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network. Biotechnol. Bioeng. 2011; 108:82–92. © 2010 Wiley Periodicals, Inc.     

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.     

Two new C3H mouse ascites tumor cell lines capable of proliferation in vivo and in suspension culture: Morphological,karyological, kinetic,and immunological properties

Summary The establishment of mouse tumor cell lines capable of proliferating in vivo and in suspension culture was undertaken. The MM-46 tumor line, initiated from primary mammary carcinoma arising in a C3H/He mouse, was maintained for over 100 generations in the peritoneal cavities of syngenic mice. At the 50th generation of the tumor suspension, cultures were initiated. The established cell lines, designated TMT-1 and TMT-2, were characterized in vitro and in vivo. The morphological finding indicated that TMT-1 and TMT-2 cells from mice closely resembled the MM-46 tumor cells. The oncogenic potential of the cultured cells was comparable to that of the original ascites tumor. The population doubling time of TMT-1 and TMT-2 cell lines was about 12 hr in mice, whereas the population doubling time of both cell lines lengthened to 20 hr in suspension culture. The increase of doubling time in culture was due to the prolongation of the G₁ period. The cell lines, TMT-1 and TMT-2, whether from culture or mice, possessed colony forming ability in soft agar medium. The colony forming ability of the cells decreased gradually through in vivo passages but it recovered upon recultivation of the cells from mouse to culture. Chromosome analysis and cytotoxicity test by anti-MM antiserum indicated that TMT-1 and TMT-2 cell lines closely resembled and had been derived from MM-46 tumor line. Therefore, it is possible to assay cell survival in vitro after in vivo experiments on these cells.     

Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy

Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion. New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations. A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function. In recent years parameters more closely related to in vivo irritancy effects such as synthesis of inflammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated. This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy. Advantages and limits of skin cell cultures are also presented. Current questions about the validation process of cutaneous irritation and corrosion are also considered.     

Chemometric evaluation of on-line high-pressure liquid chromatography in mammalian cell cultures: analysis of amino acids and glucose.

An on-line high-pressure liquid chromatography (HPLC) system capable of measuring amino acids and carbohydrates was used to study metabolism in mammalian cell culture systems. The HPLC method utilized anion-exchange chromatography followed by integrated pulsed amperometric detection. The method is capable of measuring 19 amino acids plus glucose with a complete method time of 65 min. In actual cell cultures, the method was shown to be useful for monitoring 17 amino acids plus glucose. The two amino acids that were not accurately monitored were arginine and lysine, possibly due to their elution near the void volume of the column. The HPLC system was used to study variability in metabolism across different cell culture processes, as well as the effect of glucose and glutamine limitation on a single cell culture process. Chemometric analysis was used to draw statistically meaningful conclusions from the highly correlated, multivariate data set that resulted from these experiments. Using chemometrics, variation between processes was linked to differences in uptake rates of seven amino acids. Similarly, lactate concentration, cell density, and aspartate uptake rate were linked to glucose and glutamine limitation. The effect of nutrient limitation on glutamate, alanine, and ammonium was also considered.     

Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.     

Computer-based image analysis for the automated counting and morphological description of microalgae in culture

A largely unexplored area is the application of digital image processing to counting and sizing of microalgal cells from culture. Commercial systems are available, but have not been tested, nor necessarily optimized for high speed counting and sizing of phytoplankton. The present work describes the design, construction, specifications and comparative performance of an inexpensive system optimized for counting and sizing microalgal cells. This system has been tested with cells of the picoplankton to nanoplankton size ranges (1–20 μm). The hardware was a widely available standard microcomputer, an inexpensive video camera and monitor, and a video digitization board (frame grabber). A modifiable menu-driven program (PHYCOUNT) was written and provisions made to make this program available to other workers. The program is constructed such that it can be adapted to a variety of hardware setups Video digitization boards). Comparison of growth curves for microagae revealed there were no significant differences in division rate and cell yield as assessed by the image analysis method compared to manual counts with a hemacytometer. Several hundred cells were counted routinely within 10–15 s, far exceeding the counting rate achieved by hand tally. A variable transect feature allowed sampling every nth pixel and provided a substantial increase in execution speed. More than 1000 counts can be done per day. A protocol for the use of 96-well plates of polyvinyl chloride as counting chambers contributed to the processing of large numbers of samples rapidly. Other routines developed provided subtended area, defined the coordinates of cell perimeter, and derived cell length and width. The calculation of the latter two parameters was usually done off-line as data output is in standard numerical form accessible by other programs. Experience with daily use of the PHYCOUNT program and imaging hardware reveal that the system is reliable for cell counting and sizing. The presence of bacteria in the algal cultures does not affect cell counting or sizing.     

Flow dynamics within a bioreactor for tissue engineering by residence time distribution analysis combined with fluorescence and magnetic resonance imaging to investigate forced permeability and apparent diffusion coefficient in a perfusion cell culture chamber

This study reveals that residence time distribution (RTD) analysis with pH monitoring after acid bolus injection can be used to globally study the flow dynamics of a perfusion bioreactor, while fluorescence microscopy and magnetic resonance imaging (MRI) were used to locally investigate mass transport within a hydrogel scaffold seeded or not with cells. The bioreactor used in this study is a close‐loop tubular reactor. A dispersion model in one dimension has been used to describe the non‐ideal behavior of the reactor. From open‐loop experiments (single‐cycle analysis), the presence of stagnant zones and back mixing were observed. The impact of the flow rate, the compliance chamber volume and mixing were investigated. Intermediate flows (30, 45, 60, and 90 mL min⁻¹) had no effect over RTD function expressed in reduced time (θ). Lower flow rates (5 and 15 mL min⁻¹) were associated to smaller extent of dispersion. The compliance chamber volume greatly affected the dynamics of the RTD function, while the effects of mixing and flow were small to non‐significant. An empirical equation has been proposed to localize minima of the RTD function and to predict Pe_r. Finally, cells seeded in a gelatin gel at a density of 800,000 cells mL⁻¹ had no effect over the permeability and the apparent diffusion coefficient, as revealed by fluorescent microscopy and MRI experiments. Biotechnol. Bioeng. 2011;108: 2488–2498. © 2011 Wiley Periodicals, Inc.