Application of anoxia with glucose addition for the enhanced production of hCTLA4Ig in transgenic rice suspension cell cultures

To enhance the production of hCTLA4Ig in transgenic rice suspension cell cultures, anoxic conditions were applied during the production phase. Under the anoxic conditions in sugar-depleted media, cell viability was reduced rapidly and protease activity increased compared to aerobic conditions. However, the maximum production level of hCTLA4Ig with sugar-depleted anoxic conditions was the same as that in aerobic conditions. In addition, the production of hCTLA4Ig under anoxic conditions reached a peak 2 days earlier than that in aerobic conditions. Addition of 30 mM glucose at the production phase under anoxic conditions markedly improved cell viability. A viability level over 65% could be maintained for more than 30 days. Repression of the RAmy3D promoter by residual sugar in the production of hCTLA4Ig was not observed under anoxic conditions with 30 mM glucose. In addition, the production periods of hCTLA4Ig was extended up to 30 days and the maximum production level of hCTLA4Ig under anoxic conditions was 2.1-fold higher. Therefore, anoxic conditions could be used for the enhanced production of hCTLA4Ig in transgenic rice cell cultures.     

Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures

The effects of culture media on the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and intracellular protein expression patterns were investigated in transgenic rice cell suspension cultures. Using comparative proteomic analysis, changes in the intracellular proteome in different culture media were identified. Culture media were found to be an important factor for the production of the recombinant target protein in this expression system, which was under the control of the rice α-amylase 3D (RAmy3D) promoter. In terms of hCTLA4Ig production, the N6 medium produced a 3.7-fold higher level of protein than the AA medium. In addition, the N6 medium provided better protein stability and cell viability. In the intracellular proteome analysis, we identified eight proteomes that were differentially expressed. These results could provide valuable information for the improvement of cell growth and target protein production.     

Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures

Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L⁻¹ in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.     

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l⁻¹ at day 10.     

Assessment of Recovery Medium for Production of hCTLA4Ig after Cryopreservation in Transgenic Rice Cells

A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.     

Comparative proteomic analysis for hCTLA4Ig production in transgenic rice suspension cultures using two-dimensional difference gel electrophoresis

The new technology, two-dimensional difference gel electrophoresis (2D DIGE), uses fluorescent dyes to simplify the process of detecting and matching proteins between multiple gels by allowing for the separation of up to three separate protein samples within the same gel. In this study, recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4lg) was produced in transgenic rice suspension cell cultures and the intracellular proteins were analyzed by 2D DIGE. The highest level of hCTLA4Ig (25.4 mg/L) was obtained five days after induction. The intracellular proteins expressed at both the growth and induction culture stages were separated and analyzed using DeCyder software. At least 2,218 spots were detected with two-fold thresholds and 95% confidence. We found that 29 spots increased and 20 spots decreased in their intensities during the production of recombinant hCTLA4Ig. In addition, the 2D Western blot of hCTLA4Ig revealed that this fusion protein was expressed in a variety of isoforms.     

Effective delivery of siRNA to transgenic rice cells for enhanced transfection using PEI-based polyplexes

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.     

Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig^P) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig^M), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig^P and hCTLA4Ig^M had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig^P and hCTLA4Ig^M had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-γ, but did not suppress the expression of macrophage-derived cytokines, including TNF-α and IL-1β, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig^P is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig^M.     

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.     

Skin-specifically transgenic expression of biologically active human cytoxic T-lymphocyte associated Antigen4-Immunoglobulin (hCTLA4Ig) in mice using lentiviral vector

Xenogeneic skin, especially porcine skin, has already been used to cover large wounds in clinic practice of wound care. Our previous data showed that transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic burn wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong its survival in xenogeneic wounds for coverage. Lentiviral transgenesis provides an extremely efficient and cost-effective method to produce transgenic animals. However, tissue-targeted transgenic expression of biologically functional protein by lentiviral transgenesis is rarely reported. In this work, a recombinant lentiviral vector (LV), named FKCW in this article, was constructed by inserting a skin-specific hCTLA4Ig expression cassette consisting of keratin 14 (K14) promoter, hCTLA4Ig coding sequence and an intronic fragment. Its efficacy for transgenesis and skin-specific expression of bio-active hCTLA4Ig protein was tested using mice as models. The LV FKCW was readily to be packaged and concentrated to high titres (1.287-6.254 × 10(9) TU/ml) by conventional lentivirus package system. Using eggs collected from only five mated females having been subjected to conventional super-ovulation treatment, 8 hCTLA4Ig transgenic founder mice were generated with the concentrated FKCW vector, and transgenic founder per injected and transferred egg was 6.3%, which was nearly 9-fold higher than that for DNA micro-injection with a similar transgene construct in our previous work. The lentiviral transgenic hCTLA4Ig exhibited strictly skin-specific expression at a level comparable to or even slightly higher than that of transgenic hCTLA4Ig delivered by micro-injection in a similar cassette. Lentiviral transgenic hCTLA4Ig protein remarkably suppressed human lymphocyte proliferation in vitro to a degree comparable to that of commercially purchased purified hCTLA4Ig protein with defined activity at similar concentrations. Besides, lentiviral hCTLA4Ig transgenic mouse skin grafted into rat burn wounds exhibited remarkably extended survival compared to wild-type skin of the same strain (13.8 ± 3.8 vs. 6.8 ± 3.0 days), indicating that lentiviral transgenic hCTLA4Ig did inhibit immune rejection against xenogeneic skin graft in vivo. These results laid down the foundation to further efficiently generate transgenic pigs skin-specifically expressing bio-active hCTLA4Ig by lentiviral transgenesis, and provided a demonstration that transgenic animals with tissue-targeted expression of biologically functional protein can be efficiently produced using LV.     

Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig

Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (−196 °C) after slow prefreezing in a deep freezer (−70 °C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.     

Steroid conversions by Flavobacterium dehydrogenans in two-liquid-phase systems

The production of 4-androstene-3, 17-dione by Flavobacterium dehydrogenans from androstenolone-acetate in octane/culture (v/v = 1:1) two-liquid phase systems was compared with the conversion rate in a Tween-containing medium commonly used in industry, and in a culture where no organic solvent or detergent was added. Variation of the cell density of the cultures at the moment of steroid, Tween, and/or organic solvent addition showed that the highest conversion rate in the three systems was reached with cells which were in the late exponential growth stage. The rate of 4-androstene-3, 17-dione production at the optimal cell density in the two-liquid-phase system and Tween medium were ca. 6 and 1(1/2) times as high, respectively, as in the aqueous medium without addition. The beneficial effect of octane on the rate of steroid conversion during growth could be explained by viability changes as measured by a new method to determine viability. Furthermore, our results show that a very high degree of conversion can be reached in the two-liquid-phase system [more than 98% (w/w)] which cannot be obtained in an aqueous medium. An easy way to recover the product in high purity is proposed.     

Improvement of monoclonal antibody production in hybridoma cells by dimethyl sulfoxide

Hybridoma cultures are routinely used as a source for monoclonal antibody (mAb) production necessary for preclinical evaluation. However, these cultures typically have low volumetric and specific productivities. In this article, we examined the use and the timing of addition of dimethyl sulfoxide (DMSO) as a medium additive to improve mAb production in our hybridoma clone 19 (c19) cultures. From shake flask studies, we defined the optimal DMSO concentration and time of addition for improved productivity. This timing coordinated with high cell viability and density. Hybridoma cultures treated with DMSO up to 0.3% (v/v) possessed cell densities and viabilities comparable to untreated control. We demonstrated that 0.2% (v/v) DMSO added to shake flask cultures at their maximal viable cell densities resulted in a 2-fold increase in specific mAb production. This procedure was scaleable up to 20 L Cellbags (Wave Bioreactors) with similar titer improvement. Moreover, DMSO treatment did not affect the bioactivity or glycosylation profiles of the mAb.     

A cyclical semicontinuous process for production of human alpha 1-antitrypsin using metabolically induced plant cell suspension cultures

Transgenic rice suspension cultures were utilized to produce a human therapeutic protein, recombinant alpha(1)-antitrypsin (rAAT), in a cyclical, semicontinuous operation. Recombinant protein production was induced by removing the carbon source from the cell culture medium. The transgenic rice cells secreted the rAAT into the medium, and therefore medium exchanges could be performed for consecutive growth and protein expression phases. The process consisted of three cycles over a 25-28 day period, with growth phases lasting 4-6 days each and protein expression phases lasting 2.5-5 days each. Biomass and sugar concentrations, oxygen uptake rate, cell viability, culture pH, total extracellular protein, and active rAAT were measured throughout the cyclical process. The data profiles were reproducible between separate cyclical runs where, following each induction period, cell growth and viability could be reestablished once sucrose was added back to the culture. Volumetric productivities ranged from 3 to 12 mg active rAAT/(L day) for individual cycles with overall volumetric productivities of 4.5 and 7.7 mg active rAAT/(L day).     

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8–4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid.The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53–39 mg/L per cycle during five recycling cycles.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

Culture of Isolated Single Cells from <Emphasis Type="Italic">Taxus</Emphasis> Suspensions for the Propagation of Superior Cell Populations

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4×10⁵ cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mm), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days⁻¹ was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.     

条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

Effect of oleic acid on vegetative growth of the aphid-pathogenic fungus Erynia neoaphidis

Abstract The aphid-pathogenic fungus Erynia neophidis grew on a semi-defined medium containing 16 g·1⁻¹ glucose, 3 g·1⁻¹ yeast extract and 5 g·1⁻ mycological peptone only when the medium was supplemented with low concentrations of certain fatty acids. Of these, oleic acid fulfilled the growth requirement at a concentration of 0.02% (v/v), but higher concentrations were toxic, causing complete loss of viability of cultures at a concentration of 0.2% (v/v) in liquid medium and at 4% (v/v) on solid medium. The reduced viability of the fungus in liquid culture compared to that on equivalent solid medium, and at low inoculum density compared to high inoculum density in liquid medium, is explicable in terms of this toxicity.