Production Of Suspension Cultures Research Articles

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Cell-line screening and process development for a fusogenic oncolytic virus in small-scale suspension cultures

Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108 TCID50/mL and 7.5 × 107 TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV.Key points• Cell contact-dependent oncolytic virus (OV) replicates in suspension cells.• Oncolytic potency is not encompassed during suspension cultivation.• Media composition, cell line, and MOI are critical process parameters for OV production.• The designed process is scalable and shows great promise for manufacturing clinical-grade material.

Optimization of cell growth and bacoside-A production in suspension cultures of Bacopa monnieri (L.) Wettst. using response surface methodology

Plant secondary metabolites have emerged as potential raw materials, which are used in the pharmaceutical, food, chemical, and cosmetic industries. Bacoside-A, a secondary metabolite produced by Bacopa monnieri, is known for its memory-facilitating properties. In recent years, various strategies have been developed to enhance biomass accumulation and synthesis of secondary compounds in cultures. In the present investigation, various factors affecting the production of biomass and bacoside-A in the cell suspension cultures of B. monnieri were optimized using the statistical experimental design approach. Preliminary screening by Plackett–Burman’s design revealed that among the tested factors, glucose, KNO3, KH2PO4, and inoculum density significantly influenced cell growth and bacoside-A production. Furthermore, using response surface methodology (RSM), glucose, KNO3, and KH2PO4 at a concentration of 5.67, 0.313, and 0.29%, respectively, and an inoculum density of 0.66% in basal MS medium were found to be optimal for cell growth and bacoside-A production. After optimization, the biomass yield increased about twofold (from 5.52 to 12.58 g L−1 fresh cell weight) and bacoside-A production about 1.7-fold (5.56 to 9.84 mg g−1 dry weight). The present study results show the successful application of RSM to enhance the production of biomass and accumulation of bacoside-A content in cell suspension cultures of B. monnieri.

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m−2 s−1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m−2 s−1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.

Strategies to Improve the Production of Forskolin from Hairy Root Cultures of Coleus forskohlii Briq.

The aim of this study was to elucidate the effect of elicitors and precursors on the production of forskolin from the hairy root cultures of Coleus forskohlii Briq. Hairy root cultures were established from leaf explants by infecting with Agrobacterium rhizogenes strain A4 on MS basal medium. Suspension cultures of hairy root cultures were initiated in MS medium containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L). We investigated the growth of biomass and forskolin production in suspension cultures of hairy roots. The production of forskolin was parallel to the growth of biomass. The maximum production of forskolin was observed after 5 weeks. With the objective to increase the yield of forskolin, abiotic elicitors such as salicylic acid (100 μM and 500 μM), copper sulphate (100 μM and 500 μM), methyl jasmonate (100 μM and 500 μM) and precursors such as α-ketoglutaric acid (0.2 mM and 1.0 mM), L-phenylalanine (0.2 mM and 1.0 mM) were added to hairy root cultures on different days of incubation period and evaluated their effects on production of forskolin. Elicitor, methyl jasmonate (500 μM) and the precursor, L-phenylalanine (1 mM) on day-14 addition significantly enhanced the production of forskolin over the control hairy root cultures C. forskohlii. Given forskolin’s limited commercial supply, this study provides avenues for improving the production of forskolin in the hairy root culture of C. forskohlii.

Osmotic stress induced-capsaicin production in suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili

The influence of osmotic stress on capsaicin production was investigated in cell suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili, a chili species native to Northeastern India. The sterilized seeds were germinated in Murashige and Skoog medium. Two-week-old hypocotyls were excised from in vitro germinated seedlings and implanted in MS medium containing 2, 4-dichlorophenoxyacetic acid (2 mg/l), and Kinetin (0.5 mg/l) for callus induction. Capsaicin production in the suspension cultures was significantly affected using sucrose, mannitol, and NaCl in the medium. Stoichiometric analysis with different combinations of sucrose and non-sugar osmotic agent (NaCl) showed that osmotic stress was an important factor for enhancing capsaicin production in cell suspension cultures of C. chinense. The capsaicin content of 1,644.1 μg g−1 f.wt was recorded on day 15 in cultures grown in MS medium containing 87.64 mM sucrose in combination with 40 mM NaCl. However, osmotic stress treatment at 160 mM NaCl with sucrose resulted in lowering capsaicin accumulation and separation of cell wall from their cytoplasm, under microscopic observation.

Elicitors and precursor induced effect on L-Dopa production in suspension cultures of Mucuna pruriens L.

Plant cell cultures provide an alternative means for producing secondary metabolites. The present study is an attempt to study the effect of some elicitors and precursor molecules on the production of L-Dopa (3, 4-dihydroxy-L-phenylalanine), which is a neurotransmitter precursor being used for symptomatic relief of Parkinson's disease. It is one of the most highly active allelochemicals present in Mucuna pruriens. Suspension cultures were established on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and indole acetic acid (IAA). Elicitors such as methyl jasmonate, chitin, pectin, yeast extract and also a precursor L-tyrosine at different concentrations were supplemented to the suspension cultures at different concentrations, and their effect on L-Dopa production was studied. L-Dopa produced was quantified by high performance liquid chromatography (HPLC). Compared to the control set of suspension cultures a several-fold increase in L-Dopa concentration was observed in elicitors treated and precursor fed suspension cultures. Compared to elicitor treatments precursor feeding resulted in higher concentrations of L-Dopa production.

Induced accumulation of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br.

This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata , an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly different when cells were cultivated on B5 or half-strength MS medium. However, cultivation of V. glabrata cell cultures at 25°C yielded 1.06- and 1.09-fold higher values of cell growth and 20-hydroxyecdysone content, respectively than those at 30°C. Sucrose at 30 and 40 g/L favors the production of 20-hydroxyecdysone in suspension cultures of V. glabrata . Feeding of cholesterol at 5 mg/L, as precursor for biosynthesis of 20-hydroxyecdysone, yielded 1.11-fold higher accumulation of 20-hydroxyecdysone than the control cells. Increasing of cholesterol to 10 mg/L resulted in decreased production of 20-hydroxyecdysone. Key words : Vitex glabrata, 20-hydroxyecdysone, suspension culture, cholesterol.

Significant Enhancement of Triterpenoids Production by <i>Phomopsis</i> Elicitors in Cell Suspension Cultures of <i>Betula Platyphylla Suk</i>.

58 fungal elicitors prepared from the endophytic fungi isolated from inner bark of B. platyphylla Suk. were examined to determine their effects on the growth and triterpenoid production in suspension cultures of Betula platyphylla Suk. cells. The results showed that different fungal elicitors could cause diversely stimulating effects. Among the fungal elicitors tested, BE58 stimulated the highest triterpenoids yield with 15.90 mg•L-1 and 183.72% higher than the control. The experiment also affirmed from mRNA level that triterpenoid was indeed accumulated in suspension culture of birch cells treated by BE58 fungal elicitor. Meantime BE58 was identified as Phomopsis from the morphological and molecular level.

Methyl jasmonate- and salicylic acid-induced d-chiro-inositol production in suspension cultures of buckwheat (Fagopyrum esculentum)

The aim of this work is to investigate the effects of methyl jasmonate (MeJ) and salicylic acid (SA) on d-chiro-inositol (DCI) production in buckwheat (Fagopyrum esculentum) suspension cultures. In this study, adding optimal concentrations of MeJ and SA at an appropriate time markedly increased DCI production (yield 6.141 and 5.521 mg/g DW, respectively). In addition, treatment of buckwheat cultures with a combination of 0.2 mM MeJ and 0.6 mM SA on days 0 and 9 increased the DCI yield to 7.579 mg/g DW, which was 3.726 times higher than that in the control; furthermore, the former yield was higher than that achieved by the addition of either elicitor alone. Moreover, unlike MeJ, SA did not exert a negative effect on cell growth.

Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium composition and substrate feeding

Oleandrin has been identified as the most potent antitumor ingredient of the Mediterranean herb Nerium oleander L. A strategy for optimization of medium compositions and conditions was developed for enhanced oleandrin production in suspension cultures from leaf-origin explants of Nerium oleander. The cell suspension cultures were grown in various modifications of MS medium as a basal medium. The effects of different natural extracts, plant growth substances, carbon and nitrogen sources and phosphate on the growth and oleandrin accumulation were investigated as well as effect of light, pH, shaking speed and substrate feeding. The highest oleandrin yield was obtained when the nitrogen concentration was lowered to two-thirds and the phosphate concentration increased by two-thirds of that specified in the MS medium in the presence of 3% sucrose, coconut milk, indolebutyric acid and benzyladenine in concentrations of 1 and 2 mg l−1, respectively. Lower pH and faster shaking speed favored oleandrin accumulation. Chemical feeding of progesterone and cholesterol boosted the oleandrin concentration to higher levels reaching 8.23±0.05 mg g−1 dry weight. This was about 10-fold higher than that detected in field-grown plants using the same extraction and analytic conditions, and about 24-fold higher than that determined in control cultures with regular MS medium and without precursor feeding.

Enhanced catharanthine and vindoline production in suspension cultures of <em>Catharanthus roseus</em> by ultraviolet-B light

Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation.

Optimization of Cell Growth and 20-hydroxyecdysone Production in Cell Suspension Culture of Vitex glabrata R.Br.

The effects of the cultivation media, plant growth regulators and inoculum size on the cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata R.Br. were investigated. The cell growth and 20-hydroxyecdysone formation reach the highest when cells are cultured in the Gamborg's B5 medium supplemented with 2.0 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L 2,4-D. The maximum 20-hydroxyecdysone productivity, of about 1.1 mg/L/day, was observed in the culture with 20% PCV (packed cell volume) of inoculum size. These data also show that the increment of the inoculum size to 20% PCV could increase the productivity in 7-folds.

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 x 10(6) viable cells ml(-1). At the end of the growth period glucose was completely depleted from the culture medium, but L: -lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.

Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag+) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l(-1) chitosan and 75 mu M Ag+ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l(-1), which was 3.6-fold that of the control. The glucose-6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.

Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1-5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm3 of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm-3 indolebutyric acid, 0.5 mg dm-3 kinetin and 30 g dm-3 sucrose. A 1 % CO2 supply was found beneficial for the production of cell mass; however, increasing CO2 concentration to 2.5 and 5 % decreased the biomass accumulation. CO2 enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO2 supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.

Enhancement of polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by optimization of medium compositions and feeding of sucrose

A strategy that optimization of medium compositions for maximum biomass followed by feeding of sucrose for maximum polysaccharide synthesis was developed for enhancing polysaccharide production in suspension culture of protocorm-like bodies (PLBs) of Dendrobium huoshanense C.Z. Tang et S.J. Cheng. In growth stage, the original half-strength MS medium was optimized with carbon sources, nitrogen sources and metal ion combinations. The effects of different carbon sources on PLBs growth were remarkable and sucrose at 35 g l −1 was the most suitable. Sole nitrate nitrogen of 30 mmol l −1 was the best for PLBs growth. Metal ions (Ca 2+, Fe 2+, Mn 2+ and Zn 2+) showed different influences on PLBs growth. The optimal concentration of Ca 2+, Fe 2+, Mn 2+ and Zn 2+ was 4.5 mmol l −1, 0.1 mmol l −1, 0.5 mmol l −1 and 0.06 mmol l −1, respectively. In the optimized medium (sucrose, nitrate, Ca 2+, Fe 2+, Mn 2+ and Zn 2+ concentration as described above, the other component concentration seen in half-strength MS), 33.9 g DW l −1 PLBs were harvested after 30 days of culture and biomass increase was improved 245% as compared with that in the original medium. In production stage, polysaccharide synthesis was significantly improved by the feeding sucrose. The maximum polysaccharide production (22 g l −1) was obtained in the case of 50 g l −1 sucrose feeding at day 30 of culture, which was about 109-fold higher than that in the original medium without feeding of sucrose.

Effect of Homogeneity on Cell Growth and Anthocyanin Biosynthesis in Suspension Cultures of Vitis vinifera

The instability of secondary metabolite production is a ubiquitous problem in plant cell cultures. To understand the instability, the investigation of anthocyanin accumulation in suspension cultures of Vitis vinifera, as a model system, has been initiated in our laboratory. Suspension cultures of a relatively homogeneous cell line E of V. vinifera were established by long-term cell-line selection based on the differences in their anthocyanin contents. The aggregate size of E was smaller than that of other cell lines obtained using the routine screening method. The variation coefficients of anthocyanin content in suspension cultures of E were 8.7 % in long-term subcultures and 5 % in repeated flasks. The effects of elicitation, precursor feeding, and light irradiation on biomass and anthocyanin accumulation in suspension cultures of E were investigated, and the results showed that all the variation coefficients were lower than 12 %, which indicated the importance of homogeneity on the stability of anthocyanin production in plant cell cultures. With the combined treatment of 30 μmol/L phenylalanine and 218 μmol/L methyl jasmonate in the dark in suspension cultures of E, the anthocyanin content and production in suspension cultures of E were 5.89-fold and 4.30-fold of the controls, respectively, and all the variation coefficients of biomass and anthocyanin accumulation were lower than those of the controls in five successive subcultures.

Effect of elicitation and feeding on the precursors for the production of taxanes in Taxus baccata L. suspension culture

Cell cultures of Taxus species are considered as a promising alternative source for the anticancer drug taxol (paclitaxel) and related taxanes [1, 2]. In this work we studied effect of jasmonic acid (JA) as an elicitor and 10-deacetylbaccatin III (10-DAB) and N-benzoyl-3-phenyl-isoserin (BPI) as precursors on taxanes production in suspension cultures of T. baccata L.. Baccatin III, taxol and 7-epitaxol content was measured in the medium and cells by HPLC with UV/VIS detector. The production of taxanes increased significantly after the addition of the precursors. The combination of all three treatments gave the highest yield of taxanes (45.23mg/L of media) with 10% taxol. Baccatin III content in the medium increased 292 times, taxol 24 times and 7-epitaxol 23 times compared with control cultures. In cultures fed with precursors JA, baccatin III as well as taxol and 7-epitaxol production was affected positively. In control cultures most taxanes were retained in cells (89%) and only negligible amounts were measured in the medium. When the production reached maximum, 81% of total taxanes were found in the medium. Taxol was distributed almost equally between the medium (53%) and the cells (47%). In our experiment 10-DAB was essential for the increasing of taxanes production. We presume that JA acts upon the taxol biosynthetic path between baccatin III and taxol in T. baccata suspension culture because of the relatively highest enhancement of taxol when elicited and none-elicited cultures are compared. However, the content of baccatin III was also increased after the treatment with JA [3].

Fluid mixing in shaken bioreactors: Implications for scale-up predictions from microlitre-scale microbial and mammalian cell cultures

Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation. A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are illustrated for two different cell cultures. The first involves an automated microscale process ( 1000 μ l ) for the aerobic fermentation of E. coli JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4 l scale mechanically stirred bioreactor is then demonstrated based on experiments performed at constant k L a values. The second process investigated involved antibody production in suspension cultures of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory basis for scale translation from shaken microwells ( 800 μ l ) to conical flasks (100 ml) and are indicative of results obtained in a mechanically stirred bioreactor (3.5 l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key parameters may be used to define suitable scale-up criteria for different cell cultures.