Characterization of aggregate size in Taxus suspension cell culture

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R ² > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.     

Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures

Plant cell aggregates have long been implicated in affecting cellular metabolism in suspension culture, yet the rigorous characterization of aggregate size as a process variable and its effect on bioprocess performance has not been demonstrated. Aggregate fractionation and analysis of biomass-associated product is commonly used to assess the effect of aggregation, but we establish that this method is flawed under certain conditions and does not necessarily agree with comprehensive studies of total culture performance. Leveraging recent advances to routinely measure aggregate size distributions, we developed a simple method to manipulate aggregate size and evaluate its effect on the culture as a whole, and found that Taxus suspension cultures with smaller aggregates produced significantly more paclitaxel than cultures with larger aggregates in two cell lines over a range of aggregate sizes, and where biomass accumulation was equivalent before elicitation with methyl jasmonate. Taxus cuspidata (T. cuspidata) P93AF cultures with mean aggregate sizes of 690 and 1,100 μm produced 22 and 11 mg/L paclitaxel, respectively, a twofold increase for smaller aggregates, and T. cuspidata P991 cultures with mean aggregate sizes of 400 and 840 μm produced 6 and 0.3 mg/L paclitaxel, respectively, an increase of 20-fold for smaller aggregates. These results demonstrate the importance of validating experiments aimed at a specific phenomenon with total process studies, and provide a basis for treating aggregate size as a targeted process variable for rational control strategies.     

Transformation of Althaea officinalis L. by Agrobacterium rhizogenes for the production of transgenic roots expressing the anti-HIV microbicide cyanovirin-N

The marshmallow plant (Althaea officinalis L.) has been used for centuries in medicine and other applications. Valuable secondary metabolites have previously been identified in Agrobacterium rhizogenes-generated transgenic ‘hairy’ roots in this species. In the present study, transgenic roots were produced in A. officinalis using A. rhizogenes. In addition to wild-type lines, roots expressing the anti-human immunodeficiency virus microbicide candidate, cyanovirin-N (CV-N), were generated. Wild-type and CV-N root lines were transferred to liquid culture and increased in mass by 49 and 19 % respectively over a 7 day culture period. In the latter, the concentration of CV-N present in the root tissue was 2.4 μg/g fresh weight, with an average secretion rate into the growth medium of 0.02 μg/ml/24 h. A. officinalis transgenic roots may therefore in the future be used not only as a source of therapeutic secondary metabolites, but also as an expression system for the production of recombinant pharmaceuticals.     

Variation of Telomerase Activity and Morphology in Porcine Mesenchymal Stem Cells and Fibroblasts during Prolonged in vitro Culture

The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-ß-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P < 0.05) higher in MSCs than in fibroblasts at all the passages. The PDT and cellular size slightly increased in MSCs at later passages. In contrast, fibroblasts had significantly (P < 0.05) increased PDT and cellular size, and the morphology revealed senescent-like abnormal type after passage 10. Further, the high incidence of ß-galactosidase stained cells was observed in fibroblasts compared to that of MSCs at passage 15, and cell cycle stage at G0 / G1 phase was significantly (P < 0.05) increased in the fibroblasts at 15 and 20 passages compared to that of MSCs. Based on these observations, we concluded that porcine MSCs possessed more tolerance against senescence and aging compared to fibroblasts following prolonged in vitro culture.     

Enhanced anthocyanins and resveratrol production in Vitis vinifera cell suspension culture by indanoyl-isoleucine, N-linolenoyl-L-glutamine and insect saliva

The effects of two synthetic elicitor indanoyl-isoleucine (In-Ile), N-linolenoyl-l-glutamine (Lin-Gln) and one biotic elicitor insect saliva (from Manduca sexta larvae) on plant cell cultures with respect to the induction of secondary metabolite production were investigated. Stimulated production of secondary metabolites, particularly anthocyanins in plant cells and phenolic acids in culture medium, was studied by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. In the treatments with In-Ile, the production of anthocyanins was enhanced 2.6-fold. In-Ile, Lin-Gln and saliva significantly elevated the accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol. The used elicitors did not suppress cell growth. Secondary metabolites were differently responsive to elicitation. 3-O-glucosyl-resveratrol was the predominant phenolic acid in V. vinifera cell culture, and its production was significantly stimulated by saliva, with 7.0-fold of the control level 24 h after treatment. The production of 4-(3,5-dihydroxy-phenyl)-phenol was significantly stimulated by In-Ile with 6.4-fold of the control level 24 h after treatment.     

Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of <Emphasis Type="Italic">Artemisia absinthium</Emphasis> L.

Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9 %). Moreover, the effects of 3 % repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5 %. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7 % were optimum for both classes of metabolites, respectively. Following 3 % extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.     

A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90–98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10⁶ cells/T25 flask or 1 × 10⁵ cells/cm²). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.     

Dysregulated gliotoxin biosynthesis attenuates the production of unrelated biosynthetic gene cluster-encoded metabolites in Aspergillus fumigatus

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. The gliotoxin bis-thiomethyltransferase, GtmA, attenuates gliotoxin biosynthesis in A. fumigatus by conversion of dithiol gliotoxin to bis-thiomethylgliotoxin (BmGT). Here we show that disruption of dithiol gliotoxin bis-thiomethylation functionality in A. fumigatus results in significant remodelling of the A. fumigatus secondary metabolome upon extended culture. RP-HPLC and LC–MS/MS analysis revealed the reduced production of a plethora of unrelated biosynthetic gene cluster-encoded metabolites, including pseurotin A, fumagillin, fumitremorgin C and tryprostatin B, occurs in A. fumigatus ΔgtmA upon extended incubation. Parallel quantitative proteomic analysis of A. fumigatus wild-type and ΔgtmA during extended culture revealed cognate abundance alteration of proteins encoded by relevant biosynthetic gene clusters, allied to multiple alterations in hypoxia-related proteins. The data presented herein reveal a previously concealed functionality of GtmA in facilitating the biosynthesis of other BGC-encoded metabolites produced by A. fumigatus.     

Improved production of withanolides in shoot suspension culture of Withania somnifera (L.) Dunal by seaweed extracts

The influence of Gracilaria edulis and Sargassum wightii extracts was investigated for the production of biomass and withanolides in the multiple shoot suspension culture of Withania somnifera. Supplementation of 40 % G. edulis extract in MS liquid medium for 24 h exposure time in the culture recorded the highest biomass accumulation [62.4 g fresh weight and 17.82 g dry weight (DW)] and withanolides production (withanolide A 0.76 mg/g DW; withanolide B 1.66 mg/g DW; withaferin A 2.80 mg/g DW and withanone 2.42 mg/g DW) after 5 weeks of culture, which were 1.45–1.58-fold higher than control culture. This naturally available G. edulis extract-treated multiple shoot suspension culture protocol offers a potential alternative for the optimum production of biomass and withanolides utilizing shake-flasks.     

Aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. for triptolide, wilforgine, and wilforine production

The relationships between aggregate cell types, cell growth, and the triptolide, wilforgine, and wilforine content in aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. were examined. Aggregate cells larger than 2?mm grew quickly and constituted the majority of the white aggregates. The accumulation of triptolide was strongly correlated with the size of the aggregates and the length of the culture period. The aggregates 0.5?C2?mm in diameter accumulated higher triptolide content than those with other sizes throughout the culture. However, the size of the aggregate cells did not significantly affect on the wilforgine and wilforine content. Two other kinds of aggregate cells, the brown and green aggregate cells, also formed in the suspension cultures. The smallest aggregates (0.1?C0.5?mm) had a lower biomass and growth rate and had more chloroplasts and higher alkaloid content. The results of this study can be used to improve the selection process for the mass production of triptolide, wilforgine, and wilforine from cell suspension cultures.     

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance.     

Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes

Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.     

A population balance model to modulate shear for the control of aggregation in Taxus suspension cultures

Cellular aggregation in plant suspension cultures directly affects the accumulation of high value products, such as paclitaxel from Taxus. Through application of mechanical shear by repeated, manual pipetting through a 10 ml pipet with a 1.6 mm aperture, the mean aggregate size of a Taxus culture can be reduced without affecting culture growth. When a constant level of mechanical shear was applied over eight generations, the sheared population was maintained at a mean aggregate diameter 194 μm lower than the unsheared control, but the mean aggregate size fluctuated by over 600 μm, indicating unpredictable culture variability. A population balance model was developed to interpret and predict disaggregation dynamics under mechanical shear. Adjustable parameters involved in the breakage frequency function of the population balance model were estimated by nonlinear optimization from experimentally measured size distributions. The optimized model predictions were in strong agreement with measured size distributions. The model was then used to determine the shear requirements to successfully reach a target aggregate size distribution. This model will be utilized in the future to maintain a culture with a constant size distribution with the goal of decreasing culture variability and increasing paclitaxel yields.     

Alternative carbohydrates promote differentiation of plant cells

Corn syrups have been evaluated in media for embryogenesis, androgenesis and the production of secondary metabolites from plant tissue culture. In the systems examined, higher productivity was obtained with media containing corn syrups than with comparable media containing glucose or sucrose. Corn syrup did not increase growth of unorganized cell cultures. Increased productivity therefore reflects a syrup-mediated promotion of cell differentiation. The effects of corn syrup on increasing yields of secondary metabolites were evident only after several passages in syrup-containing medium. This shows the importance of monitoring production over several passages to determine the effect of different carbon sources on secondary metabolite production. Superiority of the syrup is due primarily to the component sugars maltose and glucose. Mixtures of these sugars gave higher yields of secondary products than either sugar used alone.Abbreviations DP degree of polymerization     

Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

Water-soluble natural products from seaweed have limited potential in controlling bacterial pathogens in fish aquaculture

Seaweeds producing natural products with in vitro efficacy against bacterial pathogens offer the opportunity for therapeutic services in fish aquaculture, for example inhibiting the growth of pathogens through the direct release of bioactive metabolites into the culture environment. The red alga Asparagopsis taxiformis was used to test this hypothesis because of its effective natural products and mechanisms to release these metabolites into the surrounding environment and because it can be cultivated intensively in land-based systems. Here we quantify the release, accumulation and residence time of the major halogenated metabolites from A. taxiformis in the cultivation medium and subsequently test the in vitro bioactivity of these water-soluble metabolites against multiple strains of the pathogenic bacterium Streptococcus iniae. The two major halogenated metabolites in A. taxiformis (bromoform and dibromoacetic acid) were released into the water and reaching steady-state concentrations of 27.7?±?1.5 μg L^?1 and 4.8?±?1.3 μg L^?1, respectively, under standard cultivation conditions. This water delayed the in vitro growth of the pathogenic bacteria S. iniae but did not prevent it. Increasing the levels of A. taxiformis metabolites in the water by three orders of magnitude with aqueous extracts of biomass inhibited the growth of S. iniae, but it was also toxic to fish. In an immersion challenge experiment, intermediate non-ichthyotoxic levels of A. taxiformis metabolites were ineffective as a treatment for barramundi infected by S. iniae. The evidence suggests that there is little potential for A. taxiformis bioactive metabolites to provide therapy services to fish infected with S. iniae.     

Ecological Correlates of Ranging Behavior in Bearded Sakis (Chiropotes sagulatus) in a Continuous Forest in Guyana

Group size and the distribution and quality of food resources are among the most important determinants of primate ranging behavior. In this study, I use the framework of the ecological constraints model to assess correlates of range size of a free-ranging group of bearded sakis (Chiropotes sagulatus). Bearded sakis are among the widest ranging neotropical primates, yet the lack of data from continuous forest populations has made understanding the factors influencing such large ranges difficult. I collected data on ranging behavior and diet during 44 full-day follows over 15 mo. The focal group used a home range of ca. 1000 ha and had daily path lengths of 2.8–6.5 km (mean?=?4.0 km). Daily path length did not significantly correlate with group size, patch quality, food availability, or the spatial distribution of feeding trees. Monthly home range size significantly positively correlated with group size and patch quality. The focal group had significantly shorter paths when ripe fruit consumption was higher and had more diverse diets, visited more food patches, and used larger monthly home ranges when they consumed a higher percentage of seeds. The results of this study, combined with other recent studies of Chiropotes in continuous forest, suggest that large home ranges (approaching 1000 ha) are characteristic of the genus. Although range size may be related to group size and food patch size, I suggest nutrient mixing and the need to balance the effects of seed secondary compounds as additional explanations for the large ranges of bearded sakis.     

Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch

In this study, callus and cell suspension were induced from seedlings of licorice (G. uralensis). In addition, it was revealed that the appropriate concentration of sucrose could promote the callus growth and increase the content of polysaccharide. The methyl jasmonate (MJ) and phenylalanine (PHE) could enhance the callus growth and content of flavonoids for G. uralensis. For producing more flavonoids and polysaccharide, two-stage cultivation was performed. In the first step, 30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day of culture to enhance cell production and metabolite production. In a two-stage cultivation process, PHE (2 mM) and MJ (5 mg L^?1) were added into a 5-L balloon-type bubble bioreactor after 10 days of culture. Using a fed-batch cultivation strategy (30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day), polysaccharide production was enhanced to 1.19 g L^?1, which was 2.12-fold greater than that in batch cultivation. The flavonoids yield (55.42 mg L^?1) which was about 22 % higher than that in batch cultivation was obtained on 21st day. In a two-stage cultivation process, the polysaccharide content was increased by 1.14- and 2.12-fold compared with fed-batch cultivation and batch cultivation on 15th day. Meanwhile, total flavonoids yield (132.36 mg L^?1) on 15th day, was increased by 2.26- and 2.67-fold compared with fed-batch cultivation and batch cultivation. In conclusion, two-stage cultivation process combined with the sucrose and elicitor treatment could promote both the callus growth and the secondary metabolites accumulation.     

Neutral lipid production in <Emphasis Type="Italic">Dunaliella salina</Emphasis> during osmotic stress and adaptation

The salt-tolerant green microalga Dunaliella salina can survive both hyper- and hypo-osmotic shock. Upon osmotic shock, the cells transiently and rapidly decreased or increased in size within minutes and slowly over hours acquired their original cell size and volume. Cell size distribution differs significantly in the cultures grown in the salinity range from 1.5 to 15 % NaCl. By using Nile Red fluorescence to detect neutral lipids, it became clear that only hyper-osmotic shock on cells induced transient neutral lipid appearance in D. salina, while those transferred from 9 to 15 % NaCl stimulated the most neutral lipid accumulation. These cells grew well in 9 % NaCl, but they cannot recover a shift to 15 % NaCl and cell division is accordingly slowed down. The transient appearance of neutral lipid could be dependent on the inhibition of cell division experiencing the NaCl shift. Moreover, the effect of nutrient limitation slows down cell division and photosynthesis as a secondary result, which triggers the cells to accumulate neutral storage lipids when they entered the stationary phase, which is seen in all the batch cultures of D. salina grown in the salinity range of 3–15 %. The changes in salt concentration did not significantly influence the overall fatty acid composition in D. salina cells. Although there shows both increased amounts of total lipids and neutral lipids in the cells grown in salinity higher than 9 % NaCl, lipid productivity is however compromised by the slower cell growth rate and lower cell density under this condition.