Growth Of Suspension Cultures Research Articles

Inhibition of the mitogen activated protein kinase pathway potentiates radiation-induced cell killing via cell cycle arrest at the G2/M transition and independently of increased signaling by the JNK/c-Jun pathway.

The ability of low dose ionizing radiation (2 Gy) to modulate the activities of the mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK1) cascades in human monocytic leukemia (U937/pREP4) cells and in cells over-expressing dominant negative c-Jun (TAM67) (U937/TAM67) was investigated. Radiation exposure caused prolonged ( approximately 1 h) MAPK activations in U937 cells. In contrast, low dose irradiation weakly modulated JNK1 activity in these cells. Inhibition of the MAPK pathway by use of the specific MEK1/2 inhibitor (10 microM PD98059) in both U937/pREP4 and U937/TAM67 cells prior to radiation exposure permitted strong prolonged radiation-induced activations of JNK1. Expression of TAM67 decreased the ability of radiation to cause apoptosis compared to control transfected cells. However, combined MEK1/2 inhibition and radiation exposure in both cell types caused a large decrease in suspension culture growth and a large increase in apoptosis, when compared to either treatment alone. Reduced proliferation after combined irradiation and PD98059 treatment in both cell types correlated with prolonged cell cycle arrest in G2/M phase. Prolonged growth arrest was abolished when MEK1/2 inhibitor was removed 6 h following irradiation, which was associated with a reduction in apoptosis. The ability of MEK1/2 inhibition to cause prolonged G2/M growth arrest was reduced in U937 cells stably transfected with a p21Cip-1/WAF1 antisense construct (U937/p21AS). This data correlated with an enhancement of radiation-induced apoptosis and a reduced ability of MEK1/2 inhibition to potentiate apoptosis. Collectively our data demonstrate that inhibition of MEK1/2 function increases the radiation sensitivity of U937 cells, independently of c-Jun function, and decreases the ability of these cells to recover from the radiation-induced G2/M cell cycle checkpoint arrest. In addition, our data also demonstrate that the ability of MEK1/2 inhibition to potentiate radiation-induced cell death in U937 cells in part requires an ability of cells to express low levels of p21Cip-1/WAF1.

Rollinia mucosa cell suspension cultures : Establishment and growth conditions

Different types and concentrations of plant growth regulators were tested in order to obtain the best callus and cell suspension culture growth conditions of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Picloram was shown to be the most efficient for induction and production of friable calluses, independent of the concentration used. Cellular morphology and viability, fresh and dry weights, pH and medium sugar concentration were determined for cell suspension cultures. Dissimilation curves were used for the characterization of the growth of cell suspension cultures. Picloram provided the most rapid growth and produced the highest biomass, with little variation in morphology (differentiated cells). It also provided the highest dissimilation, when compared with cell suspension cultures maintained in media with 2,4-D or NAA + BA + GA3. Stable cell suspension cultures can be established in MS medium supplemented with 20.8 μM picloram.

Screening of transformed insect cell lines for recombinant protein production.

Nine insect cell lines were evaluated for their potential as host systems for recombinant protein production using a new expression vector permitting the continuous high-level expression of secreted glycoproteins by transformed insect cells (Farrell et al., 1998). As a means of preliminary screening, all nine insect cell lines were transfected with the green fluorescence protein. Growth in static and suspension culture was then examined as a further method of screening. On the basis of their transfection efficiencies and cell growth characteristics, five insect cell lines, Bm5, High Five, IPLB-LdFB, IZD-MB-0503, and Sf-21, were selected for stable transformation to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). These five cell lines were stably transformed using an antibiotic resistance scheme and evaluated as a polyclonal population. Increasing the antibiotic concentration was found to cause not only a decrease in the specific growth rate but also an increase in the specific protein production rate and final GM-CSF concentration. The transformed High Five cells exhibited by far the greatest specific protein production rate of 5.1 x 10(-)(6) microgram/(cell.h), resulting in the highest final GM-CSF concentration of 22.8 mg/L when grown in static culture. One cloned High Five cell line produced a GM-CSF concentration of 46 mg/L in static culture and 27 mg/L in suspension culture.

Broth rheology, growth and metabolite production of Beta vulgaris suspension culture: a comparative study between cultures grown in shake flasks and in a stirred tank

Cells of Beta vulgaris have the ability to grow in a stirred tank under an impeller tip speed as high as 95.3 cm seg −1. Comparing this system with cultures performing in shake flasks, a decrease of the cell concentration, betalains production, and growth rate was observed. However, the kinetic profiles of aggregates size and cellular viability were practically the same. The cultures carried out in the fermentor showed a major accumulation of extracellular arabinogalactoprotein and polysaccharide, which is an indication of the cell response to hydrodynamic stress. These extracellular molecules produced a considerable change in the rheology of cell-free medium. This change in the rheology can be playing an important role in the reduction of the actual hydrodynamic stress during cultivation.

Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design

In order to develop a serum-free (SF) medium for the production of erythropoietin (EPO) by suspension culture of recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett–Burman design was adopted. A basal medium was prepared by supplementing Iscove’s modified Dulbecco’s medium (IMDM) with Fe(NO 3) 3 9H 2O, CuCl 2 and ZnSO 4 7H 2O which are generally contained in SF medium formulations. Insulin, transferrin and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, glutamate, serine, methionine, phosphatidylcholine, hydrocortisone and pluronic F68 were identified as positive determinants for cell growth. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth in suspension culture. An EPO titer in this optimized SF medium was 79% of that in IMDM supplemented with 5% dialyzed fetal bovine serum (dFBS). Furthermore, the in vitro and in vivo biological activities of EPO produced in the SF medium were comparable to those produced in the serum-supplemented medium. Taken together, the results obtained here show that a Plackett–Burman design facilitates the development of SF media for the production of EPO by suspension culture of rCHO cells.

Carrot cell growth response in a stimulated ultrasonic environment

Carrot cells were exposed in suspension to 28 kHz continuous wave ultrasound. Exposure durations were from 2 to 40 s at the beginning of the cell suspension culture, so as to encompass the first decade of decreased or increased survival at each action time. In our experiment, we used a spectrophotometer to assay cell growth by means of measurement directly the absorbance (ABS) change in suspension culture. The experimental results indicated whether it would have been a positive or negative influence on plant cell growth in suspension culture with ultrasound exposed time varying; Meanwhile, we got two shake modes, a reciprocal shaker and a rotary shaker, that had almost same influences on plant cell growth under assigned conditions.

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.

Chemotherapeutic agents used in the treatment of childhood malignancies have direct effects on growth plate chondrocyte proliferation.

Short stature is one of the most well recorded long term sequelae for adult survivors of childhood malignancies. It has become increasingly apparent that cytotoxic chemotherapy, as well as craniospinal irradiation, has a major impact on growth, but there are virtually no studies which explore the mechanisms by which these cytotoxic drugs affect growth. We have used an in vitro system to investigate the direct effects of a range of chemotherapeutic agents on the proliferative responses of rat tibial growth plate chondrocytes, both in suspension and monolayer culture. The glucocorticoids and purine anti-metabolites reduced chondrocyte proliferation both in monolayer and suspension cultures and this resulted from an increase in cell doubling times with a concomittant reduction in the numbers of S phase cells. DNA damaging agents (e.g. actinomycin-D) were also able to reduce chondrocyte proliferation, both in monolayer and suspension culture. This, however, was the result of a cell cycle arrest and subsequent cell death. In our studies, methotrexate had no significant effect on the proliferative responses of the chondrocytes either in monolayer or suspension culture. These results indicate direct effects of a range of chemotherapeutic agents on the proliferative responses of growth plate chondrocytes. Both cytostatic and cytotoxic effects were observed although the impact of either the potential loss of cells from the proliferative pool during chondrocyte differentiation, or the reduction in the rate of chondrocyte turnover on long bone growth remains to be elucidated.

Tip growth in xylogenic suspension cultures ofZinnia elegans L.: Implications for the relationship between cell shape and secondary-cell-wall pattern in tracheary elements

The relationship between cell expansion, cortical microtubule orientation, and patterned secondary-cell-wall deposition was investigated in xylogenic cell suspension cultures ofZinnia elegans L. The direction of cell expansion in these cultures is pH dependent; cells elongate at pH 5.5–6.0, but expand isodiametrically at pH 6.5–7.0. Contrary to our expectations, indirect immunofluorescence revealed that cortical microtubules are oriented parallel to the long axis in elongating cells. Pulse labeling of the walls of isolated cells with the fluorochrome Tinopal LPW demonstrated that xylogenic Zinnia mesophyll cells elongate by tip growth in culture. These results confirm that cortical microtubules in developing tracheary elements reorient before bundling to form transverse cortical microtubule bands. This rearrangement may allow the secondary cell wall pattern to conform to cell shape, independent of the direction in which the cell was expanding prior to reorientation.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry ( Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO 4, NO 3, NH 4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d −1 based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest that the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Sucrose starvation causes a loss of immunologically detectable pyrophosphatase and V-ATPase in the tonoplast of suspension-cultured tobacco cells

Summary The effect of sucrose starvation on logarithmically growing suspension cultures of tobacco BY-2 cells has been investigated by immunogold electron microscopy with antisera against pyrophosphatase (PPase) and V-ATPase. In normal cells PPase is located at the plasma and tonoplast membranes, whereas V-ATPase is restricted to the tonoplast. After 32 h of growth in the absence of exogenous sucrose, both PPase and V-ATPase polypeptides are no longer detectable in the tonoplast by this method. By contrast, plasma membrane-bound PPase appears to be unaffected by sucrose deficiency. These results are discussed in terms of a down-regulation of electrogenic pumps in the tonoplast.

Internal Zinc Accumulation Is Correlated with Increased Growth in Rice Suspension Culture

A high zinc concentration of 520 μm, approximately 100 times that used most often in standard plant tissue culture media, was found to be superior in liquid callus cultures of japonica rice, increasing growth to 146% compared with standard N6 medium. At the same time, the internal zinc concentration increased 40 times in fast growing cells; soluble protein doubled, and free amino acids decreased. Under zinc-free conditions the cultures slowed in growth, and several free amino acids such as aspartic acid, glutamic acid, asparagine, and glutamine accumulated. We suggest that zinc acts as a direct regulatory factor in inducing auxin activity, but not auxin levels, making high internal zinc accumulation mandatory if high auxin concentrations are required as in rice callus cultures.

Glucose and Stress Independently Regulate Source and Sink Metabolism and Defense Mechanisms via Signal Transduction Pathways Involving Protein Phosphorylation.

In higher plants, sugars are required not only to sustain heterotrophic growth but also to regulate the expression of a variety of genes. Environmental stresses, such as pathogen infection and wounding, activate a cascade of defense responses and may also affect carbohydrate metabolism. In this study, the relationship between sugar- and stress-activated signal transduction pathways and the underlying regulatory mechanism was analyzed. Photoautotrophically growing suspension culture cells of Chenopodium rubrum were used as a model system to study the effects of the metabolic regulator D-glucose and of different stress-related stimuli on photosynthesis, sink metabolism, and defense response by analyzing the regulation of mRNAs for representative enzymes of these pathways. Glucose as well as the fungal elicitor chitosan, the phosphatase inhibitor endothall, and benzoic acid were shown to result in a coordinated regulatory mechanism. The mRNAs for phenylalanine ammonia-lyase, a key enzyme of defense response, and for the sink-specific extracellular invertase were induced. In contrast, the mRNA for the Calvin cycle enzyme ribulose bisphosphate carboxylase was repressed. This inverse regulatory pattern was also observed in experiments with wounded leaves of C. rubrum plants. The differential effect of the protein kinase inhibitor staurosporine on mRNA regulation demonstrates that the carbohydrate signal and the stress-related stimuli independently activate different intracellular signaling pathways that ultimately are integrated to coordinately regulate source and sink metabolism and activate defense responses. The various stimuli triggered the transient and rapid activation of protein kinases that phosphorylate the myelin basic protein. The involvement of phosphorylation in signal transduction is further supported by the effect of the protein kinase inhibitor staurosporine on mRNA levels.

Effect of temperature and its shift on growth and anthocyanin production in suspension cultures of strawberry cells

Temperature effect on growth and anthocyanin production in the cultured strawberry, Fragaria ananassa cv Shikinari, was investigated at a temperature range of 15–35°C. Cultivation temperatures have a pronounced effect on cell growth, anthocyanin content and production. Maximum growth was found at 30°C, however, the lower temperature induced an increased level of anthocyanin content within the examined temperatures. As a result, the maximum anthocyanin production was obtained at 20°C. The culture condition was changed step-wise by a temperature shift after 3 days from 30 to 20°C for the rest of the cultivation. In this process, anthocyanin production of 270 mg/l on day 9 was increased 1.8, 3 and 4-fold over that of cultures at 20, 25 and 30°C, respectively.

Polyamine Sparing May Be Involved in the Prolongation of Cell Division Due to Inhibition of Phenylpropanoid Synthesis in Cytokinin-Starved Soybean Cells

Effects on growth, mostly of an inhibitory nature, have been attributed to phenolic compounds in vivo and in vitro. This suggests that l-α-aminooxy-β-phenylpropionic acid (l-AOPP), a competitive inhibitor of phenylalanine ammonia-lyase (PAL), the enzyme controlling the first step in phenylpropanoid synthesis, might stimulate growth in soybean suspension cultures (Glycine max, cv. Acme). The promotive effect of l-AOPP, measured as an increase in cell number, was more clearly detected in the growth-limiting condition of cytokinin starvation. At least one more cell division cycle was completed in the presence of l-AOPP before growth by division ceased and growth continued by expansion only. Phenolic acids are known to conjugate with polyamines, modulating the free levels of these plant growth substances. Thus, the effect of l-AOPP on the titers of free and conjugated polyamines (putrescine, spermidine, and spermine) was investigated by high performance liquid chromatography in the course of cytokinin starvation. An increased level of free putrescine was detected in the presence of l-AOPP relative to controls, especially in the initial period before growth became restricted to cell expansion. The decrease in free putrescine associated with the cessation of cell division was temporarily delayed, suggesting that an interaction between phenolic acids and polyamines is involved in the mechanism of growth promotion by l-AOPP.

Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 µM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM.

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained.

Possible correlation between increased vigour and chitinase activity expression in tobacco

Nicotiana tabacum (tobacco) plants with several-fold increased chitinase enzyme activity levels were obtained through transformation with a Zea mays (maize) chitinase cDNA clone, Ch2, under the control of the CaMV 35S promoter. There was a linear correlation (r=0.74, P=0.009) between the endochitinase activity and the weight of homozygous seedlings grown in the greenhouse in some experiments. Progeny seedlings of self-pollinated regenerants (heterozygous for Ch2) grown for 18 d in Petri plates could be separated into vigorous and non-vigorous categories based on size, which were predicted to be 2n or 1n and null, respectively, for Ch2. Testing showed that the visually estimated vigour of these progeny co-segregating for Ch2 and nptII generally correlated with resistance to kanamycin and chitinase activity. There was also a correlation between growth and chitinase activity in suspension cultures derived from transgenic and wild-type when measured after 20 d. Twelve-day-old wild-type and transformed seedlings grown in soil under culture room conditions in closed plastic containers were visibly taller and had higher seedling weights than their counterparts grown under mist in the greenhouse. Likewise, the chitinase activity in the cell wall-bound proteins of the faster growing seedlings was more than double that of the greenhouse-grown seedlings. While these studies do not always demonstrate the correlation between vigour and chitinase activity, overall, they do support the hypothesis that chitinases have a role in plant growth.

CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins

Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. (c) 1996 John Wiley & Sons, Inc.

Somatic embryogenesis of pepper in bioreactors: a study of bioreactor type and oxygen-uptake rates

Somatic embryogenesis of pepper, Capsicum annuum var. Ace, was performed in an airlift bioreactor and a magnetically stirred hanging-stirrer-bar bioreactor, each with 1.81 working volume. All stages of embryogenesis, from growth of embryogenic suspension cultures to embryo maturation, were performed in the bioreactor as a series of drain-and-fill batches, keeping the cells and embryos in the bioreactor all the time. When two bioreactors were compared in terms of percentage embryogenesis and visually observed quality of mixing, under different rates of aeration and stirring, the performance of the magnetically stirred bioreactor was better. The effects of inoculum type and inoculum level on the percentage embryogenesis were also investigated. Under the optimum conditions, embryogenesis was 98%, with 57 embryos/ml. Oxygen-uptake rates of cultures in different stages of embryogenesis were different, the highest being in the embryogenic suspension culture and the lowest during embryo maturation.