Development of transfection and high-producer screening protocols for the CHOK1SV cell system

To date, the FDA has approved 18 monoclonal antibody (MAb) therapeutic drugs with targets ranging from asthma and rheumatoid arthritis to leukemia. Many of these approved products are produced in Chinese hamster ovary cells (CHO) making CHO a significant and relevant host system. We studied the applicability of CHOK1SV cells as a potential host cell line for MAb production in terms of timelines, achievable titers, transfectant stability, and reproducibility. CHOK1SV, developed by Lonza Biologics, is a suspension, protein-free-adapted CHOK1-derivative utilizing the glutamine synthetase (GS) gene expression system. CHOK1SV expresses the GS enzyme endogenously; thus, positive transfectants were obtained under the dual selection of methionine sulfoximine (MSX) and glutamine-free media. We examined outgrowth efficiencies, specific productivities, and achievable batch titers of three different IgG MAbs transfected into CHOK1SV. Reducing the MSX concentration in the initial selection medium resulted in a decreased incubation time required for transfectant colonies to appear. Specific productivities of “high-producers” ranged between 11 and 49 pg/c/d with batch titers ranging from 105 to 519 mg/L. Transfectant stability and the effects of MSX also were investigated, which indicated that the addition of MSX was necessary to maintain stable MAb production. Cell growth was stable regardless of MSX concentration.     

Capsaicin formation in p-fluorophenylalanine resistant and normal cell cultures ofCapsicum frutescens and activity of phenylalanine ammonia lyase

Callus cultures ofCapsicum frutescens capable of producing a maximum of 53 μg capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000 μM to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000 μM registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 μM PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoid pathway in resistant cell cultures was studied and compared with normal cell cultures to understand its role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.     

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

In Lonza Biologics' GS Gene Expression System?, recombinant protein‐producing GS–CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine‐free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER‐resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS–CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS–CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb‐producing GS–CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight‐fold and the concentration in harvest medium by two‐fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process‐driven method for increasing mAb productivity from GS–CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17–25, 2017     

Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.     

In vitro propagation for the conservation of a rare medicinal plant <Emphasis Type="Italic">Justicia gendarussa</Emphasis> Burm. f. by nodal explants and shoot regeneration from callus

Justicia gendarussa is a valuable medicinal plant and various parts of this plant are pharmaceutically used for the treatment of different diseases. In vitro regeneration of shoot buds was obtained from culture of nodal cuttings as well as shoot regeneration from callus. The nodal cuttings differed in shoot proliferation in terms of percentage of explants that responded and average shoot length with various concentrations (4.4, 8.9, 13.3, 17.7, 22.2 μM) of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron. In all treatments, one shoot was invariably present. Optimum 87% of cultures responded with an average shoot length of 4.4 cm on Murashige and Skoog (MS) medium supplemented with 17.7 μM BA. Callus was induced from the mature leaf segments on MS medium supplemented with Kn (4.7, 13.9, 23.2 μM) alone or in combination with 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.3 μM, 4.5 μM). Optimum callus induction (78%) was obtained on MS medium supplemented with 14 μM Kn and 4.5 μM 2, 4-D. When the callus was subcultured on MS medium fortified with BA (8.9, 17.7, 26.6 μM) or Kn (9.3, 18.6, 27.9 μM) alone or in combination with α naphthalene acetic acid (NAA; 2.7, 5.4 μM), shoot regeneration was obtained. The highest response (92%) was observed on MS medium containing 17.7 μM BA and 5.4 μM NAA. On this medium, an average number of 12.2 shoots were obtained per responding callus. The shoots obtained from callus and nodal cuttings were rooted with a frequency of 73% on MS medium augmented with 9.8 μM indole-3-butyric acid. The rooted shoots were successfully transplanted to soil and sand mixture (1:1) with 90% survival rate. The protocol standardized for shoot proliferation and regeneration in J. gendarussa from nodal cuttings and leaf-derived callus is suitable for micropropagation and conservation of this essential medicinal plant.     

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K _d values of 2.6 ± 0.1 μM for HBA and 1.9 ± 0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min⁻¹ and 61 ± 1 min⁻¹, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω−1- and ω−2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω−2-hydroxynonyloxybenzoic acid and ω−2,ω−4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω−2- and 0.5 mM ω−1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min⁻¹, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min⁻¹.     

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L⁻¹ of dry cell weight (OD₆₀₀ = 120) within 24 h. A plasmid DNA yield of 100 mg L⁻¹ (1.7 mg g⁻¹ cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L⁻¹) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L⁻¹ was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10–12 g L⁻¹ (OD₆₀₀ = 25–30) and plasmid yields of 5–8 mg L⁻¹ (approximately 0.7 mg g⁻¹ cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (μ) from 0.69 h⁻¹ to 0.13 h⁻¹ and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. Received 22 March 1996/ Accepted in revised form 20 September 1996     

Modulating zucchini cotyledon plate meristem activity by interactions between the cycline-dependent kinase inhibitor roscovitine and cytokinins

The inhibitors of cytokinin N-glucosylation are known to influence the growth of some plant objects including cotyledons. The use of the plate meristem of zucchini cotyledon as an experimental system allowed us to study for the first time the way in which the changes in the cell division are integrated in this growth reaction. Roscovitine, a potent inhibitor of cytokinin N-glucosylation and cycline-dependent kinases, did not show to have an effect on the meristem activity when applied in 100 μM to cultivated zucchini cotyledons, and acted as an inhibitor in concentrations higher than 400 μM. A 200 μM roscovitine stimulated both palisade cell division and growth. In different seed batches, 400 μM roscovitine acted as a stimulator or an inhibitor. A much stronger stimulating effect on growth and cell division was observed after application of benzyladenine (BA, 10 μM). In contrast to BA, roscovitine provoked a formation of principally flat lamina. In combined treatments, it lowered the stimulating effect of BA; 400 μM roscovitine combined with BA severely suppressed the growth and division activity. This cellular behavior and changes in cotyledon growth could be due to the roscovitine-provoked changes in endogenous cytokinin levels via the inhibition of cytokinin N-glucosylation. Roscovitine-caused stimulation of cell growth and division is stronger in the marginal meristem than that registered in central regions of the cotyledon blade. In this region it also changed the pattern of cell division and lowered the adhesion between the clusters, which enhanced the appearance of local ruptures of the cotyledon edges. The first palisade layer of the plate meristem of cultured zucchini cotyledons, the natural mono-layer of proliferating palisade cells, may be used for screening the inhibitors of cycline-dependent kinases and cytokinin N-glucosylation with regard to their effects on cell division and growth.     

Production of selected baculoviruses in newly established lepidopteran cell lines

Summary One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID₅₀ assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×10⁶ TCID₅₀/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×10⁶ TCID₅₀/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×10⁶TCID₅₀/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×10⁶TCID₅₀/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses. All programs and services of the USDA Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status or handicap.     

In vitro regeneration in <Emphasis Type="Italic">Dierama erectum</Emphasis> Hilliard

The genus Dierama comprises plants with a potential to be developed as ornamentals. D. erectum seeds were decontaminated and germinated on 1/10th strength Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) media without plant growth regulators or sucrose. In an experiment investigating the effects of 6-benzyladenine (BA), meta-Topolin (mT), kinetin (KIN) and zeatin (Z) with or without α-naphthaleneacetic acid (NAA), the highest shoot number per hypocotyl (4.20 ± 0.51) was obtained from MS medium supplemented with 1.0 μM Z after 8 weeks. This was followed by a combination of 2.0 μM KIN and 2.0 μM NAA with 3.67 ± 0.81 shoots per explant. BA treatments produced 3.20 ± 0.22 shoots per hypocotyl explant when 2.0 μM was combined with 1.0 μM NAA, while mT gave 3.09 ± 0.99 shoots per explant when 2.0 μM mT was combined with 2.0 μM NAA. Adventitious shoot regeneration was optimised when shoots were grown under a 16-h photoperiod at 100 μmol m⁻² s⁻¹ on MS medium supplemented with 1.0 μM BA. This resulted in an average of 12.73 ± 1.03 shoots per hypocotyl explant. Various concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation of this species. Plants rooted successfully after 8 weeks on MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) and had an average root number of 2.73 ± 0.40. After 2 months of acclimatisation, plants had formed corms. The largest corms (of diameter 0.45 ± 0.03 cm) were produced in plants pre-treated with 0.5 μM IBA. The highest plant survival percentage of 73% was also associated with this treatment.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of protocorm-like bodies in <Emphasis Type="Italic">Cattleya</Emphasis>

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.     

Leucine and spermidine enhance shoot differentiation in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Enhanced numbers of multiple shoots were induced from shoot tip explants of cucumber. The effects of amino acids (leucine, isoleucine, methionine, threonine, and tryptophan) and polyamines (spermidine, spermine, and putrescine) along with benzyladenine (BA) on multiple shoot induction were investigated. A Murashige and Skoog (MS) medium containing a combination of BA (4.44 μM), leucine (88 μM), and spermidine (68 μM) induced the maximum number of shoots (36.6 shoots per explant) compared to BA (4.44 μM) alone or BA (4.44 μM) with leucine (88 μM). The regenerated shoots were elongated on the same medium. Elongated shoots were transferred to the MS medium fortified with BA (4.44 μM), leucine (88 μM), and putrescine (62 μM) for root induction. Rooted plants were hardened and successfully established in soil with a 90% survival rate.     

Production of psoralen by in vitro regenerated plants from callus cultures of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

Psoralea corylifolia is an attractive, endangered annual producing various bioactive compounds of medical importance. This plant contributes to Indian pharmaceutical and cosmetic industries for the production of commercial medicines, Ayurvedic skin care ointments and soap. The influence of various plant growth regulators (PGRs) and additives on high-frequency rapid adventitious shoot regeneration from transverse thin cell layer (tTCL) hypocotyl explants of P. corylifolia was investigated. Organogenic callus was obtained in tTCL hypocotyl explants on Murashige and Skoog (1962) medium supplemented with 15 μM naphthaleneacetic acid (NAA) and 3 μM benzylaminopurine (BA). The highest adventitious shoot regeneration (107.5 shoots per explant) was achieved in culture when transferred to half-strength solid MS medium. The regenerated shoots were rooted and the plantlets successfully acclimatized in moistened (1/8-MS basal salt solution with 3 μM indole-3-butyric acid (IBA), 1 μM 2-isopentenyladenine (2iP) and 100 mg l⁻¹ Bavistin (BVN)); garden soil, farmyard soil and sand (2:1:1, v/v/v). The acclimatized plants produced flowers in the growth chamber. When planted in the field these plants set viable seed. The psoralen content in different tissues of ex vitro and naturally-grown plants was determined by high-performance liquid chromatography (HPLC). The highest psoralen content was recorded in seeds from naturally-grown (6.48 μg g⁻¹ DW) and ex vitro plants (6.46 μg g⁻¹ DW). This system can be used for rapid mass propagation of P. corylifolia, for conservation strategies, and to produce phytomedicines.     

In vitro propagation of Bambusa nutans Wall. ex Munro through axillary shoot proliferation

This communication describes for the first time an efficient and reproducible protocol for large-scale multiplication of Bambusa nutans. Nodal segments collected from field-grown clumps and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM benzylaminopurine (BA) and 2.32 μM kinetin (Kin) gelled with 0.2% gelrite yielded 80% aseptic cultures with 100% bud-break. The in vitro-formed shoots obtained after bud-break were successfully multiplied in MS liquid medium supplemented with 13.2 μM BA, 2.32 μM Kin, and 0.98 μM indole-3-butyric acid (IBA). Sub-culturing of shoots every 3 weeks on fresh multiplication medium yielded a consistent proliferation rate of 3.5-fold. Shoot clusters containing three to five shoots were successfully rooted with 100% success on half-strength MS liquid medium supplemented with 9.8 μM IBA, 2.85 μM indole-3-acetic acid (IAA), 2.68 μM naphthaleneacetic acid (NAA), and 3% sucrose. Plantlets grown in vitro were acclimatized and subsequently transferred to the field. Inter-simple sequence repeat analysis has confirmed the genetic uniformity of the tissue-cultured plants up to 27 passages.     

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H₂O₂, either alone or together. Cells were pretreated with medium containing 1, 10, or 100 μM equol for 1 h prior to the addition of 1 mM H₂O₂ for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H₂O₂ caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 μM) but not high concentrations of equol (10 μM) inhibited cell damage, while 100 μM equol caused more serious damage than H₂O₂ alone. Pretreatment with 1 μM equol had no effect on cell viability, while pretreatment with 10 and 100 μM equol significantly decreased cell viability in a dose-dependent manner. Compared with H₂O₂ alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 μM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H₂O_2, except for an obvious increase in GSH-Px activity caused by 10 μM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H₂O₂, while pretreatment with high-dosage equol shows a synergistic effect with H₂O₂ in inducing cell damage.     

FeedER: a feedback-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates

With the advent of modern genetic engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and bioprocess engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into d-glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium glutamicum, Pichia pastoris, and Escherichia coli. Moreover, the comparative analysis of a C. glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial bioprocess development at the screening stage.

     

Batch, fed-batch, and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.     

Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8–2.9 up to 4.0–4.4 μm. The diameter of the hyphal elements remained constant, around 4 μm, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations.     

Somatic embryogenesis and plant regeneration from root sections of <Emphasis Type="Italic">Allium schoenoprasum</Emphasis> L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10 μM. The highest frequencies of callus induction were achieved on media with 5 μM 2,4-D in combination with 5 μM TDZ or 10 μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1 μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5 μM 2,4-D in combination with 10 μM BA or 10 μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5 μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.