Search

This study was aimed to determine the effects of different concentrations of L-phenylalanine (0.25, 0.50 and 1.00 mM L-Phe) applied to adventitious root cultures for different culture durations on root growth and production of tropane alkaloids and phenolics in Hyoscyamus niger. For this purpose, adventitious roots obtained from petiole explants of in vitro seedlings were used as plant materials. After adventitious roots were cultured in liquid Murashige and Skoog (MS) medium, L-Phe was applied to the adventitious roots. Then the roots were harvested on the 1st, 3rd, and 7th day of the applications, fresh and dry root weights, root growth indexes were analysed. It was also examined the contents of tropane alkaloids (hyoscyamine and scopolamine) and phenolics (gallic acid, catechin, epicatechin, vanillin, cinnamic acid, rosmarinic acid, p-coumaric, o-coumaric acid, rutin, caffeic acid, ferulic acid, chlorogenic acid, and quercetin) by HPLC in both adventitious roots and culture medium. As a result, according to root growth parameters data, day*application interaction was not found statistically significant. The maximum scopolamine and hyoscyamine amounts were detected in the cultures with the application of 0.50 mM L-Phe for 3 days. In terms of phenolics, 0.50 mM and 1.00 mM L-Phe application with 1st and 3rd days were selected as the most appropriate L-Phe applications and culture duration. Consequently, it has been determined that in vitro secondary metabolite production may be increased in H. niger root cultures with the selection of appropriate L-Phe concentrations and culture durations. Key Message: L-phenylalanine is an effective and inexpensive application for the production of secondary metabolites in adventitious root cultures of Hyoscyamus niger with the correct concentration and appropriate culture duration. [ABSTRACT FROM AUTHOR]

Sweet potato (Ipomoea batatas [L.] Lam.) is an important food crop that is cultivated and consumed worldwide, and its nutritional value makes it especially important in developing countries. However, sweet potato plants produced using conventional vegetative propagation can be prone to several diseases, thereby hindering crop yield. Therefore, micropropagation techniques are useful for producing higher quality plant material. The aim of the present study was to determine the optimal medium formulation for the axillary shoot multiplication of single-node 'Abees' explants. The influences of cytokinins (6-benzyladenine [BA], kinetin, thidiazuron, and zeatin), gibberellic acid, phenylacetic acid, indole-3-butyric acid, silver nitrate, and oxalic acid, culture duration, and medium salt strength were investigated. Optimal proliferation was observed when shoots were cultured on half-strength MS medium that was supplemented with 2 mg L-1 BA and 100 mg L-1 oxalic acid for 2 wk, followed by culture on half-strength MS medium without plant growth regulators for 4 wk. In this medium, the greatest number of shoots (3.1) and total number of nodes (16.1) per explant were observed. All (100%) of the regenerated shoots were rooted and acclimatized, with a survival rate of 96% when transferred to ex vitro conditions and no morphological abnormalities. [ABSTRACT FROM AUTHOR]

ABSTRACT In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells. Biotechnol. Bioeng. 2015;112: 2172-2184. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

BACKGROUND Currently used mathematical models for animal cell growth and metabolism cannot simulate changing culture pH and its effect on metabolism, and hence cannot accurately model cultures lacking pH control. Animal cell cultures are however widely cultured at a small scale without pH control, e.g. for clone screening during cell line development. A mathematical model for animal cell growth without pH control is developed and applied to design optimal conditions for clone screening without pH control. RESULTS The mathematical model successfully simulates published data for suspension CHO culture in shake flasks. It is further used to simulate clonal heterogeneity and predict relative clone performance in different culture conditions. Predictions of correlation coefficients between clone titers in batch and fed batch screening conditions and pH controlled fed batch agree with published values. Fed batch mode is predicted to give better screening outcomes than batch, but only for clones able to consume lactate. Simulations reveal that culture duration of the screening assay has a large effect on the screening outcome and optimal durations differ based on the ability or inability of clones to consume lactate. CONCLUSION This study presents a tool for optimal design of screening assay for clone selection without pH control. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Objective: To determine whether the preantral follicles in adult ovaries can generate developmentally competent oocytes after in vitro culture. Design: Prospective, animal-model study. Setting: Gamete and Stem Cell Biotechnology Laboratory, Seoul National University, Seoul, Korea. Animal(s): B6CBAF1 mice. Intervention(s): Preantral follicles collected from 8-week-old mice were cultured in vitro. Main Outcome Measure(s): Follicle development, embryogenesis, and embryonic stem cell characterization. Result(s): A mean of 50.3 preantral follicles were retrieved from one adult animal, which is significantly less than the number (88.7 follicles) retrieved from a prepubertal female. Extension of the culture period greatly improved oocyte maturation; increased follicular growth to the pseudo-antral (89%–91% vs. 32%) or mature oocyte stage (65%–77% vs. 13%) was observed after 12 or 13 days of culture compared with 9 days of culture. Blastocyst formation after parthenogenesis was detected in only one case; in comparison, the use of IVF yielded a large number of embryos that developed into blastocysts. A mean of 14.7 intrafollicular oocytes per animal were produced after 13 days of culture, and 41% of those developed into blastocysts after IVF. Embryonic stem cell–like colonies were established by subculturing the inner cell mass cells from the blastocysts. Conclusion(s): Developmentally competent oocytes can be generated by culturing adult preantral follicles. These results may help increase the feasibility of follicle culture systems. [Copyright &y& Elsevier]

Our past studies revealed that feeding the basal frond portion of fresh Saccharina japonica kelp (BS) and the sporophyll of fresh Undaria pinnatifida (SU) during May–July for 2 months can effectively improve the gonad quality of Mesocentrotus nudus from a barren. In the present study, M. nudus were collected from a barren and fed BS, SU or no food during May–July. We investigated the temporal changes in gonad size, color and texture and the contents of free amino acids in the gonads of M. nudus every 3 weeks during May–July to identify the appropriate culturing duration. The protein, carbohydrate, and total and free amino acid contents in each kelp thallus portion were examined at each time point. The gonad quality of sea urchins fed each portion for 9 weeks was evaluated by the chefs of sushi restaurants with three or two Michelin stars in Tokyo with a sensory test. The results of the sensory test indicated that the gonads of sea urchins fed BS or SU are of high quality, the same as those served in the sushi restaurants. Feeding the sea urchins BS improved gonad hardness in 3 weeks, gonad bitterness in 6 weeks, and gonad size, color and sweetness in 9 weeks. Feeding SU improved gonad hardness and taste in 3 weeks and gonad size and color in 6 weeks. These results indicate that the use of the sporophyll enables the culture duration to shorten to 6 weeks for the production of high-quality gonads. The sweet-tasting alanine contents in the gonads of sea urchins fed BS and SU increased, coinciding with the high glutamic acid content in BS and high alanine content in SU. As nutrients in the frond of U. pinnatifida are translocated and accumulate in the sporophyll from March and May, hastening the start of U. pinnatifida sporophyll feeding would further shorten the culture duration. • Mesocentrotus nudu s were fed Undaria sporophyll or basal frond portion of Saccharina. • Gonads of sea urchins fed each portion for 9 weeks were evaluated as high quality. • Feeding Saccharina basal frond took 9 weeks to improve gonad size and taste. • Feeding Undaria sporophyll improved the whole gonad quality in 6 weeks. • Use of Undaria sporophyll shortened the culture duration. [ABSTRACT FROM AUTHOR]

Objective: To develop and test a simple, feasible approach to improve spindle assembly and developmental competence of human in vitro matured oocytes by parthenogenetic activation in maturation medium. Design: Prospective, randomized study. Setting: University research laboratory-based assisted reproductive technology laboratory. Patient(s): Four hundred thirty-two patients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles. Intervention(s): In vivo failed-to-mature oocytes from the ICSI cycles were divided into four groups according to differing duration after the extrusion of first polar body (0–1, 2–3, 4–5, and 8–9 hours). Oocytes spindles in each group were immunostained for α-tubulin and chromosomes, and observed by confocal microscopy. Main Outcome Measure(s): Rate from pronuclear stage to blastocyst, embryo grading at the eight-cell and blastocyst stages, and spindle assembly. Result(s): There was a statistically significantly higher rate of development at the eight-cell and blastocyst stages in the 2- to 3-hour and 4- to 5-hour groups. The grading results at the eight-cell and blastocyst stages also showed that the proportion of embryos of high quality was similar among the 2- to 3-hour and 4- to 5-hour groups, and in these groups it was statistically significantly higher than the 0- to 1-hour and 8- to 9-hour groups. The results of immunofluorescence demonstrated that there was a statistically significantly higher rate of normal spindle assembly in the 2- to 3-hour and 4- to 5-hour groups. Conclusion(s): Optimal timing of maturation benefits the development of competence of in vitro matured oocytes by promoting normal spindle assembly. [ABSTRACT FROM AUTHOR]

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process., (Copyright © 2011 Wiley Periodicals, Inc.)