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Suspension Cultures (suspension + culture)
Kinds of Suspension Cultures Selected AbstractsOxidative Burst in Suspension Culture of Taxus cuspidataInduced by a Laminar Shear Stress in Short-TermBIOTECHNOLOGY PROGRESS, Issue 2 2004Rong-Bin Han Generation of active oxidative species induced by shear stress in suspension cultures of Taxus cuspidata was investigated in a Couette-type shear reactor. It was found that T. cuspidata cells respond to a shear rate of 95 s,1 with oxidative bursts. Their triphasic characteristics in 6 h were similar in both intracellular H2O2 production and extracellular O2,, production. Additionally, inhibition studies with diphenylene iodonium and azide suggested that the key enzyme responsible for oxidative bursts under the shear rate of 95 s,1 is primarily NADPH oxidase and the contribution of peroxidase for oxidative bursts was less. Investigation of the relationship between active oxidative species and defense responses induced by the shear stress indicated that the O2,, burst may account for the change of membrane permeability, and the H2O2 burst plays an important role in inducing secondary metabolites such as the activation of phenylalanine ammonia lyase enzyme and phenolic accumulation. Furthermore, oxidative bursts elicited by the shear rate of 95 s,1 were suppressed by treatment with suramin, nifedipine, and neomycin prior to the shear stress treatment, suggesting that G-protein, Ca2+ channel, and phospholipase C are involved in the signal pathway for oxidative bursts induced by the shear stress. A model is proposed to explain the oxidative burst in cultured T. cuspidata cells challenged with the shear stress. [source] Comparison of Growth and Recombinant Protein Expression in Two Different Insect Cell Lines in Attached and Suspension CultureBIOTECHNOLOGY PROGRESS, Issue 4 2001R. A. Taticek Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPL,-Sf21-AE) and Trichoplusia ni (Tn 5,-1,4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150,160 rpm were necessary for maximum growth of suspended Tn 5,-1,4 cells compared to 125,150 rpm for Sf-21 cells. An inoculum size of 5 × 105 cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for ,-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5,-1,4 cells are 2,4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of ,-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5,-1,4 cells produced 2.6,4.4 and 2.7,3 times more ,-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the ,-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of ,-galactosidase by Tn 5,-1,4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production. [source] Isolation and Initial Characterization of 132 -Hydroxychlorophyll a Induced by Cyclohexanedione Derivatives in Tobacco Cell Suspension CulturesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2000Jing-Ming Wang ABSTRACT This paper reports that a new photobleaching compound, 2-(2-chloro-5-propoxycarbonylphenyl)aminomethylidene-5,5-dimethyl-cyclohexane-1,3-dione (RWH-21), stimulates accumulation of 132 -hydroxychlorophyll a in cultured tobacco cells. This was shown based on isolation of 132 -hydroxychlorophyll a from pigment extracts of cultured tobacco cells by diode-array HPLC and subsequent fast atom bombardment mass spectrometry analysis. 132 -Hydroxychlorophyll a rapidly accumulates in tobacco cells both in the light and dark in the presence of RWH-21 (50 ,M). Analysis of 132 -hydroxychlorophyll a formation in tobacco cells indicates that 132 -hydroxychlorophyll a is rapidly accumulated within 20 h incubation time both in the dark and light. Although the amount of 132 -hydroxychlorophyll a is continuously increased in the dark, the amount of 132 -hydroxychlorophyll a decreased remarkably in the light after 20 h incubation. Analysis of 132 -hydroxychlorophyll a formation and lipid peroxidation by determination malondialdehyde in tobacco cells suggests that RWH-21-induced 132 -hydroxychlorophyll a has the potential to cause a photodynamic action in cultured tobacco cells. [source] A Cyclical Semicontinuous Process for Production of Human ,1 -Antitrypsin Using Metabolically Induced Plant Cell Suspension CulturesBIOTECHNOLOGY PROGRESS, Issue 2 2005Melody M. Trexler Transgenic rice suspension cultures were utilized to produce a human therapeutic protein, recombinant ,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). [source] Improved Paclitaxel and Baccatin III Production in Suspension Cultures of TaxusmediaBIOTECHNOLOGY PROGRESS, Issue 3 2002Rosa M. Cusidó A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L,1) and baccatin III (2.56 mg L,1) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 ,g g,1 FW). When the elicitor was added together with mevalonate (0.38 mM) and N -benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two-stage culture for cell suspension was carried out using a 5,l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L,1 of paclitaxel and 56.03 mg L,1 of baccatin III were obtained after 8 days of culture in the production medium. [source] Effect of Elicitation on Growth, Respiration, and Nutrient Uptake of Root and Cell Suspension Cultures of HyoscyamusmuticusBIOTECHNOLOGY PROGRESS, Issue 2 2002Edgard B. Carvalho The elicitation of Hyoscyamus muticus root and cell suspension cultures by fungal elicitor from Rhizoctonia solani causes dramatic changes in respiration, nutrient yields, and growth. Cells and mature root tissues have similar specific oxygen uptake rates (SOUR) before and after the onset of the elicitation process. Cell suspension SOUR were 11 and 18 ,mol O2/g FW·h for non-elicited control and elicited cultures, respectively. Mature root SOUR were 11 and 24 ,mol O2/g FW·h for control and elicited tissue, respectively. Tissue growth is significantly reduced upon the addition of elicitor to these cultures. Inorganic yield remains fairly constant, whereas yield on sugar is reduced from 0.532 to 0.352 g dry biomass per g sugar for roots and 0.614 to 0.440 g dry biomass per g sugar for cells. This reduction in yield results from increased energy requirements for the defense response. Growth reduction is reflected in a reduction in root meristem (tip) SOUR, which decreased from 189 to 70 ,mol O2/g FW·h upon elicitation. Therefore, despite the increase in total respiration, the maximum local oxygen fluxes are reduced as a result of the reduction in metabolic activity at the meristem. This distribution of oxygen uptake throughout the mature tissue could reduce mass transfer requirements during elicited production. However, this was not found to be the case for sesquiterpene elicitation, where production of lubimin and solavetivone were found to increase linearly up to oxygen partial pressures of 40% O2 in air. SOUR is shown to similarly increase in both bubble column and tubular reactors despite severe mass transfer limitations, suggesting the possibility of metabolically induced increases in tissue convective transport during elicitation. [source] Growth and movement of secondary plasmodia of Plasmodiophora brassicae in turnip suspension-culture cellsPLANT PATHOLOGY, Issue 1 2006T. Asano Growth of secondary plasmodia of the clubroot pathogen Plasmodiophora brassicae was studied in dual culture of P. brassicae and turnip suspension cells. Suspension culture of P. brassicae -infected turnip cells was achieved by using P. brassicae -infected callus in Murashige and Skoog medium supplemented with 0·1 mg 2,4-D L,1 and 0·02 mg kinetin L,1. The shape of secondary plasmodia in suspension cells was spherical-to-subspherical. A few young plasmodia divided and became numerous spherical, small plasmodia which eventually formed a plasmodial cluster. The plasmodia fused and became vegetative plasmodia. Infected cells were significantly larger than noninfected cells. Secondary plasmodia moved within transformed turnip suspension host cells by cytoplasmic streaming of the host cells. Secondary plasmodia divided in synchrony with the transformed turnip cells. [source] Uptake of nicotine from suspension culture of Nicotiana tabacum by molecularly imprinted polymersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2010Mohamed Salaheldin A. Abdelkader Abstract Objectives The aim was to use molecularly imprinted polymers (MIPs) for the selective recovery of nicotine in plant cell cultures. MIPs can selectively uptake nicotine from suspension cultures of N. tabacum, and therefore may be useful for improving levels of secondary metabolites in plant cell cultures. Methods Suspension cultures of N. tabacum were initiated from callus and maintained in liquid Murashige and Skoog (MS) media containing 3% w/v sucrose, 0.1 mg/l ,-naphthaleneacetic acid acid (NAA) and 0.25 mg/l kinetin. Tween 80 at 1% was used for permeabilisation of cell cultures. Pre-weighed XAD-2 and two types of synthesized polymers, MIPs (A and B with one and two functional monomers, respectively) and corresponding non-imprinted polymers (NIPs), A and B, were introduced aseptically into the permeabilised suspension cultures of N. tabacum, the nicotine contents of polymers were determined by gas chromatography and the adsorption yield of polymers were determined. Key findings Cell cultures of N. tabacum accumulated nicotine alkaloid intracellularly in varying levels, 6.8,14.9 mg/l fresh weight. MIPs were able to uptake 50,70% of released nicotine in suspension cultures of N. tabacum, whereas XAD-2 recovered only 30,40%. The total levels of accumulated nicotine were enhanced up to 20 mg/l by simultaneous use of Tween 80 and MIPs. Conclusions The findings indicate the potential use of MIPs to uptake nicotine from suspension cultures of N. tabacum, and increase productivity of secondary metabolites in plant cell cultures. [source] Influence of Fungal Elicitors on Production of Ajmalicine by Cell Cultures of Catharanthus roseusBIOTECHNOLOGY PROGRESS, Issue 1 2002Ajay Namdeo Suspension cultures of Catharanthus roseus ( C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A.niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T.viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A.niger, F. moniliforme, and T. viride. The maximum ajmalicine production (75 ,g g,1 dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 ,g g,1 DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 ,g g,1 DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 ,g g,1 DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis. [source] Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function,HEPATOLOGY, Issue 2 2009Colleen M. Brophy The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. Conclusion: Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker-formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct. (HEPATOLOGY 2009.) [source] Overexpression of BAD preferentially augments anoikisINTERNATIONAL JOURNAL OF CANCER, Issue 2 2003Masashi Idogawa Abstract BAD is a BH3-only protein, and its proapoptotic activity is negatively regulated by serine phosphorylation. Here, we show that overexpression of BAD preferentially augments anchorage loss,induced apoptosis (anoikis). Gene transfer,mediated BAD overexpression alone did not induce apoptosis in attached MDCK cells but strongly augmented apoptosis when cells were cultured in suspension. In contrast, overexpression of another BH3-only protein, BID, displayed much lower augmentation of anoikis, suggesting a preferential contribution of BAD to anoikis. During suspension culture, unphosphorylated BAD was gradually increased and targeted to the mitochondria. Cotransfection of BAD with constitutively active Akt cDNA strongly inhibited this change. In contrast, the increase of unphosphorylated BAD was not significantly inhibited by several phosphatase inhibitors or cotransfection with a dominant negative calcineurin cDNA, implying that the increase may be mainly due to a decrease of serine kinase activity, such as that of Akt. Similar results were observed in COS-7 cells, suggesting that BAD overexpression can increase sensitivity of anchorage-dependent cancer cells to anoikis. Thus, we propose that BAD can serve as a valuable gene therapeutic molecule to inhibit carcinoma progression. © 2003 Wiley-Liss, Inc. [source] Supercritical CO2 extraction of accumulated capsidiol from biotic elicitor-activated Capsicum annuum L fruit tissuesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2005ur Salg Abstract This work investigates the supercritical CO2 extraction of capsidiol from pepper fruit tissues activated with Alternaria alternate (Fr) Keissler suspension culture as a biotic elicitor. Capsidiol production in the fruit tissue was markedly increased by the treatment with a biotic elicitor and reached its maximum level after 4 days of elicitation. The effects of separation parameters such as temperature, pressure, supercritical solvent flow rate, particle diameter and also initial capsidiol concentration were investigated on solubility, initial extraction rate and extraction yield. The optimal extraction conditions were obtained at the temperature of 40 °C, the pressure of 400 bar, the supercritical CO2 flow rate of 2 cm3 min,1, and the average particle diameter of 116 µm. The results showed that the ratio of the supercritical CO2 extraction yield to the organic solvent extraction yield was changed from 84 to 97 wt-% depending on the initial capsidiol concentration. Copyright © 2004 Society of Chemical Industry [source] Uptake of nicotine from suspension culture of Nicotiana tabacum by molecularly imprinted polymersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2010Mohamed Salaheldin A. Abdelkader Abstract Objectives The aim was to use molecularly imprinted polymers (MIPs) for the selective recovery of nicotine in plant cell cultures. MIPs can selectively uptake nicotine from suspension cultures of N. tabacum, and therefore may be useful for improving levels of secondary metabolites in plant cell cultures. Methods Suspension cultures of N. tabacum were initiated from callus and maintained in liquid Murashige and Skoog (MS) media containing 3% w/v sucrose, 0.1 mg/l ,-naphthaleneacetic acid acid (NAA) and 0.25 mg/l kinetin. Tween 80 at 1% was used for permeabilisation of cell cultures. Pre-weighed XAD-2 and two types of synthesized polymers, MIPs (A and B with one and two functional monomers, respectively) and corresponding non-imprinted polymers (NIPs), A and B, were introduced aseptically into the permeabilised suspension cultures of N. tabacum, the nicotine contents of polymers were determined by gas chromatography and the adsorption yield of polymers were determined. Key findings Cell cultures of N. tabacum accumulated nicotine alkaloid intracellularly in varying levels, 6.8,14.9 mg/l fresh weight. MIPs were able to uptake 50,70% of released nicotine in suspension cultures of N. tabacum, whereas XAD-2 recovered only 30,40%. The total levels of accumulated nicotine were enhanced up to 20 mg/l by simultaneous use of Tween 80 and MIPs. Conclusions The findings indicate the potential use of MIPs to uptake nicotine from suspension cultures of N. tabacum, and increase productivity of secondary metabolites in plant cell cultures. [source] MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 5 2006Nicole Poulsen Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non-selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co-transformation allowing for the simultaneous introduction of a non-selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana -specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms. [source] A Suspension Culture Method for the Rapid Mass Culture of Cistella japonica MyceliumJOURNAL OF PHYTOPATHOLOGY, Issue 9 2006T. Yamanobe Abstract Different methods were investigated for the rapid mass culture of Cistella japonica by using water extracts of some nutritional sources. In an agar culture test, there was little difference in mycelial growth in water extracts of wheat bran, rice bran and potato. In suspension culture with wheat bran extract, which is easily and cheaply available, the mycelium of C. japonica increased seven times more than that in agar culture after a month's incubation. C. japonica from suspension culture was pathogenic to Chamaecyparis obtusa. These results suggest that suspension culture in water extract of wheat bran can be adopted for the rapid mass culturing of C. japonica for use in inoculation tests. [source] Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°CLIVER INTERNATIONAL, Issue 3 2003Alan J. Wigg Abstract: Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 °C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 °C were also able to maintain normal Na + and K + ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers. [source] Stable transgene expression in human embryonic stem cells after simple chemical transfectionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2009Jun Liu In this study we used plasmid-based vectors to investigate the transcriptional activities of three commonly used promoters in transient and stable transfection of MEL-1, a human embryonic stem (ES) cell line, using ExGen500, Fugene HD, and Lipofectamine. We demonstrated that cytomegalovirus (CMV), phosphoglycerate kinase (PGK) and human elongation factor-1, (EF1,) promoters all resulted in robust activity of a reporter gene in MEL-1 ES cell transient transfections regardless of the transfection reagent. Stable transfection outcomes varied, depending on the promoter and the transfection reagent used in the study. The phenomenon of transgene silencing was observed, most notably with the CMV vector, with which no positive stably transfected clones were obtained. Of the methods used in the study, Fugene HD resulted in the highest stable transfection rate, estimated by antibiotic selection, with plasmids containing genes under the control of the EF1, or PGK promoters. Stably transfected cells maintained typical hES cell morphology, with immunostaining exhibiting expression of the hES cell markers: Oct4, SSEA4, Tra-1-60, and Tra-1-81. Further, embryoid bodies formed by suspension culture retained reporter gene expression. Following injection into immunodeficient mice, the transfected cell lines showed robust formation of teratomas with cell types representative of the three germ layers. Mol. Reprod. Dev. 76: 580,586, 2009. © 2008 Wiley-Liss, Inc. [source] Isolation and culture of embryonic stem cells from porcine blastocystsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Ming Li Abstract This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7,9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent. Mol. Reprod. Dev. 65: 429,434, 2003. © 2003 Wiley-Liss, Inc. [source] Photodynamic Treatment of the Dermatophyte Trichophyton rubrum and its Microconidia with Porphyrin Photosensitizers,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2004Threes G. M. Smijs ABSTRACT The application of photosensitizers for the treatment of fungal infections is a new and promising development within the field of photodynamic treatment (PDT). Dermatophytes, fungi that can cause infections of the skin, hair and nails, are able to feed on keratin. Superficial mycoses are probably the most prevalent of infectious diseases in all parts of the world. One of the most important restrictions of the current therapeutic options is the return of the infection and the duration of the treatment. This is especially true in the case of infections of the nail (tinea unguium) caused by Trichophyton rubrum, an anthropophilic dermatophyte with a worldwide distribution. Recently, we demonstrated that 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester were excellent photosensitizers toward T. rubrum when using broadband white light. This study demonstrates the photodynamic activity of these photosensitizers with red light toward both a suspension culture of T. rubrum and its isolated microconidia. The higher penetration depth of red light is important for the PDT of nail infections. In addition, we tested the photodynamic activity of a newly synthesized porphyrin, quinolino-[4,5,6,7-efg]-7-demethyl-8-deethylmesoporphyrin dimethylester, displaying a distinct peak in the red part of the spectrum. However, its photodynamic activity with red light toward a suspension culture of T. rubrum appeared to be only fungistatic. Sylsens B was the best photosensitizer toward both T. rubrum and its microconidia. A complete inactivation of the fungal spores and destruction of the fungal hyphae was found. In studies into the photostability, Sylsens B appeared to be photostable under the conditions used for fungal PDT. A promising result of this study is the demonstration of the complete degradation of the fungal hyphae in the time after the PDT and the inactivation of fungal spores, both with red light. These results offer the ingredients for a future treatment of fungal infections, including those of the nail. [source] Cation-induced superoxide generation in tobacco cell suspension culture is dependent on ion valencePLANT CELL & ENVIRONMENT, Issue 11 2001T. Kawano Abstract There have been many reports suggesting the involvement of reactive oxygen species (ROS), including superoxide anion (O2.,), in salt stress. Herein, direct evidence that treatments of cell suspension culture of tobacco (Nicotiana tabacum L.; cell line, BY-2) with various salts of trivalent, divalent and monovalent metals stimulate the immediate production of O2., is reported. Among the salts tested, LaCl3 and GdCl3 induced the greatest responses in O2., production, whereas CaCl2 and MgCl2 showed only moderate effects; salts of monovalent metals such as KCl and NaCl induced much lower responses, indicating that there is a strong relationship between the valence of metals and the level of O2., production. As the valence of the added metals increased from monovalent to divalent and trivalent, the concentrations required for maximal responses were lowered. Although O2., production by NaCl and KCl required high concentrations associated with hyperosmolarity, the O2., generation induced by NaCl and KCl was significantly greater than that induced simply by hyperosmolarity. Since an NADPH oxidase inhibitor, diphenyleneiodonium chloride, showed a strong inhibitory effect on the trivalent and divalent cation-induced generation of O2.,, it is likely that cation treatments activate the O2., -generating activity of NADPH oxidase. [source] Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymesBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2000L.H. Cohen Abstract Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC50 values: 0.2,8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug,drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6,-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S -mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1,-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O -dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 µM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC50 values around 200 µM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC50 value: 4 µM). Using the assay of testosterone 6,-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC50 values of about 200 µM and a little more by C and A (IC50 around 100 µM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug,drug interaction is considered. Copyright © 2000 John Wiley & Sons, Ltd. [source] Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010T. Holland Abstract Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10,20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8,mg,L,1 over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N -glycan analysis. Biotechnol. Bioeng. 2010;107: 278,289. © 2010 Wiley Periodicals, Inc. [source] BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Gregory J. Cost Abstract Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX - and BAK -deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330,340. © 2009 Wiley Periodicals, Inc. [source] Scalable production of adeno-associated virus type 2 vectors via suspension transfection,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Joon Young Park Abstract Vectors derived from adeno-associated virus type 2 (AAV2) are promising gene delivery vehicles, but it is still challenging to get the large number of recombinant adeno-associated virus (rAAV) particles required for large animal and clinical studies. Current transfection technology requires adherent cultures of HEK 293 cells that can only be expanded by preparing multiple culture plates. A single large-scale suspension culture could replace these multiple culture preparations, but there is currently no effective co-transfection scheme for generating rAAV from cells in suspension culture. Here, we weaned HEK 293 cells to suspension culture using hydrogel-coated six-well culture plates and established an efficient transfection strategy suitable for these cells. Then the cultures were gradually scaled up. We used linear polyethylenimine (PEI) to mediate transfection and obtained high transfection efficiencies ranging from 54% to 99%, thereby allowing efficient generation of rAAV vectors. Up to 1013 rAAV particles and, more importantly, up to 1011 infectious particles were generated from a 2-L bioreactor culture. The suspension-transfection strategy of this study facilitates the homogeneous preparation of rAAV at a large scale, and holds further potential as the basis for establishing a manufacturing process in a larger bioreactor. © 2006 Wiley Periodicals, Inc. [source] Uptake and biotransformation of 2,4,6-trinitrotoluene (TNT) by microplantlet suspension culture of the marine red macroalga Portieria hornemanniiBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Octavio Cruz-Uribe Abstract Microplantlets of the marine red macroalga Portieria hornemannii efficiently removed the explosive compound 2,4,6-trinitrotoluene (TNT) from seawater. Photosynthetic, axenic microplantlets (1.2 g FW/L) were challenged with enriched seawater medium containing dissolved TNT at concentrations of 1.0, 10, and 50 mg/L. At 22°C and initial TNT concentrations of 10 mg/L or less, TNT removal from seawater was 100% within 72 h, and the first-order rate constant for TNT removal ranged from 0.025 to 0.037 L/gFW h under both illuminated conditions (153 µE/m2s, 14:10 LD photoperiod) and dark conditions. Two immediate products of TNT biotransformation, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dintrotoluene, were identified in the liquid culture medium, with a maximum material balance recovery of 29 mole%. Only trace levels of these products and residual TNT were found within the fresh cell biomass. Removal of TNT by P. hornemannii microplantlets at initial concentrations of 1.0 or 10 mg/L did not affect the respiration rate. At an initial TNT concentration of 10 mg/L, net photosynthesis decreased towards zero, commensurate with the removal of dissolved TNT from seawater, whereas at an initial TNT concentration of 1.0 mg/L, the net photosynthesis rate was not affected. © 2005 Wiley Periodicals, Inc. [source] Suspension Culture Process of MethA Tumor Cell for the Production of Heat-Shock Protein Glycoprotein 96: Process Optimization in Spinner FlasksBIOTECHNOLOGY PROGRESS, Issue 6 2007Ya-Jie Tang Heat-shock proteins (HSPs) act like "chaperones", making sure that the cellapos;s proteins are in the right shape and in the right place at the right time. Heat-shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat-shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat-shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat-shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat-shock protein gp96. Initial glucose concentration had a significant effect on the heat-shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 × 105 cells/mL and the maximal heat-shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat-shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 × 105 cells/mL and the maximal heat-shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Not only the cell density but also the production and productivity of heat-shock protein gp96 attained in this work are the highest reported in the culture of MethA tumor cell. This work offers an effective approach for producing heat-shock protein glycoprotein 96 from the cell culture process. The fundamental information obtained in this study may be useful for the efficient production of heat-shock protein by animal cell suspension culture on a large scale. [source] Improved Paclitaxel and Baccatin III Production in Suspension Cultures of TaxusmediaBIOTECHNOLOGY PROGRESS, Issue 3 2002Rosa M. Cusidó A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L,1) and baccatin III (2.56 mg L,1) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 ,g g,1 FW). When the elicitor was added together with mevalonate (0.38 mM) and N -benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two-stage culture for cell suspension was carried out using a 5,l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L,1 of paclitaxel and 56.03 mg L,1 of baccatin III were obtained after 8 days of culture in the production medium. [source] Contrasting in vitro effects for the combination of fludarabine, cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (FLAG) compared with daunorubicin and Ara-C in P-glycoprotein-positive and P-glycoprotein-negative acute myeloblastic leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2000Y. Higashi It has been suggested that the FLAG remission induction regimen comprising fludarabine (F-ara), cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (G-CSF) may be capable of overcoming P-glycoprotein (P-gp)-related multidrug resistance (MDR) in patients with acute myeloblastic leukaemia (AML). We have investigated the in vitro response of P-gp-positive and -negative AML clones to FLAG and compared this with their response to treatment with Ara-C and daunorubicin (DNR). Twenty-four cryopreserved samples from patients with AML were studied using a flow cytometric technique for the enumeration of viable (7-amino actinomycin D negative) cells. Samples consisted of 12 P-gp-positive and 12 P-gp-negative cases, as measured by the MRK16 antibody. The results were analysed by calculating the comparative drug resistance (CDR), i.e. the percentage cell death caused by Ara-C + DNR subtracted from the percentage cell death, caused by FLAG after 48 h incubation in suspension culture. P-gp-positive clones were shown to have a significantly higher CDR than P-gp-negative clones (P = 0·001). Furthermore, a significant positive correlation (r2 = 0·40, P < 0·01) was found between P-gp protein expression and CDR. However, P-gp function, measured using cyclosporin modulation of rhodamine 123 (R123) uptake, was not associated with the CDR, demonstrating that there are other properties of P-gp, besides its role in drug efflux, that modulate the responsiveness of AML blasts to chemotherapy. These results are consistent with a potential benefit for FLAG in P-gp-positive AML, but not P-gp-negative AML, compared with standard anthracycline and Ara-C therapy. [source] Biotransformation of n -hexadecane by cell suspension cultures of Cinchona robusta and Dioscorea compositaENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2001Carolina Vega-Jarquin Abstract This manuscript evaluates the phytotoxicity and biotransformation of n -hexadecane as well as peroxidase activity and cytochrome P450 concentration in microsomes for cell suspension cultures of Cinchona robusta and Dioscorea composita. Phytotoxicity was evaluated based on viability and growth. Cell cultures were exposed to a 2 and 4% (v/v) dose of n -hexadecane. The biotransformation of n -hexadecane was determined based on labeled recovery in polar, nonpolar, and cell residue fractions after cell culture extraction during exponential cell growth phase and stationary phase. Differences were observed in accumulation of label during cell growth phase and stationary phase for the cells of the two plants. Differences also were observed between phases for label in polar and nonpolar fractions. Thin-layer chromatography determined labeled intermediates and some were identified. The activity of peroxidase and concentration of cytochrome P450 was lower in C. robusta than in controls and greater in D. composita than in controls. In vitro biotransformation was not successful. [source] Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloidsFEBS JOURNAL, Issue 8 2002Irina Gerasimenko Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (N,-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation. [source] | |||||||||||||||||||||||||||||||||||||