Cell density influences the effect of celecoxib in two carcinoma cell lines.

It has been reported that when ovarian carcinoma cell lines are exposed to various concentrations of celecoxib, a COX-2 inhibitor, cell growth is decreased in a dose dependant manner. To examine further the effect of celecoxib, different cell densities of two carcinoma cell lines were exposed to various concentrations of celecoxib. LNCAP prostate and CAOV3 ovarian carcinoma cells were obtained from the American Type Culture Collection and maintained in Rosewell Park Memorial Institute 1640 and Dulbeceo's modified Eagle's medium, respectively. Each cell line was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotic-antimycotic solution, and placed in a humidified atmosphere containing 5% CO2 at 37 degrees C. After each cell line reached a confluency of 70-80%, 1,000, 2,000, 3,000, 5,000, 7,000 and 10,000 cells/well were seeded in 96 well plates in 100 microl medium/per well for 24 h. Each cell line was exposed to the same concentrations of celecoxib (10-100 microM) at each cell density for 72 h. Cell growth was assessed using a tetrazolium conversion assay. A significant decrease compared to controls was observed in cell growth at each cell density of LNCAP and CAOV3 cells plated with >or=30 microM and >or=50 microM celecoxib, respectively. When the cell growth curves were compared for each cell density at the same concentration of celecoxib, a significant decrease in cell growth was observed when LNCAP cells were plated at 10,000 cells/well and exposed to 10-100 microM celecoxib. At a cell density >or= 5,000 LNCAP cells/well, the inhibitory effect of celecoxib was less. Similarly, a significant decrease in cell growth was observed in CAOV3 cells plated at 1,000 cells/well compared to other cell numbers plated at the same drug concentrations. At a cell density of > 5,000 CAOV3 cells/well, the inhibitory effect of celecoxib was significantly less compared to other cell densities at the same concentration. We observed a more sensitive decrease in cell growth in both carcinoma lines studied at a cell density of 1,000 cells/well with exposure to 10-100 microM celecoxib. Both carcinoma cell lines were less sensitive at a cell density of 5,000 cells/well. Our results suggest that the inhibitory effect of celecoxib may be affected by cell density. Therefore, careful attention must be paid to determining the appropriate cell density for cytotoxicity studies.     

Isber annual meeting program biodiversity and international regulations May 4–7, 2003 Adam’s Mark Hotel,Philadelphia, PA

It has been reported that when ovarian carcinoma cell lines are exposed to various concentrations of celecoxib, a COX-2 inhibitor, cell growth is decreased in a dose dependant manner. To examine further the effect of celecoxib, different cell densities of two carcinoma cell lines were exposed to various concentrations of celecoxib. LNCAP prostate and CAOV3 ovarian carcinoma cells were obtained from the American Type Culture Collection and maintained in Rosewell Park Memorial Institute 1640 and Dulbeceo's modified Eagle's medium, respectively. Each cell line was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotic-antimycotic solution, and placed in a humidified atmosphere containing 5% CO₂ at 37° C. After each cell line reached a confluency of 70–80%, 1000, 2000, 3000, 5000, 7000 and 10,000 cells/well were seeded in 96 well plates in 100?µl medium/per well for 24 h. Each cell line was exposed to the same concentrations of celecoxib (10–100?µM) at each cell density for 72 h. Cell growth was assessed using a tetrazolium conversion assay. A significant decrease compared to controls was observed in cell growth at each cell density of LNCAP and CAOV3 cells plated with ≥30?µM and ≥50?µM celecoxib, respectively. When the cell growth curves were compared for each cell density at the same concentration of celecoxib, a significant decrease in cell growth was observed when LNCAP cells were plated at 10,000 cells/well and exposed to 10–100?µM celecoxib. At a cell density?≥?5000 LNCAP cells/well, the inhibitory effect of celecoxib was less. Similarly, a significant decrease in cell growth was observed in CAOV3 cells plated at 1000 cells/well compared to other cell numbers plated at the same drug concentrations. At a cell density of?>?5000 CAOV3 cells/well, the inhibitory effect of celecoxib was significantly less compared to other cell densities at the same concentration. We observed a more sensitive decrease in cell growth in both carcinoma lines studied at a cell density of 1000 cells/well with exposure to 10–100?µM celecoxib. Both carcinoma cell lines were less sensitive at a cell density of 5000 cells/well. Our results suggest that the inhibitory effect of celecoxib may be affected by cell density. Therefore, careful attention must be paid to determining the appropriate cell density for cytotoxicity studies.     

Frankia菌种保藏

对用4种方法保藏的Frankia菌,进行了培养物存活、形态及其固氮活性的检测.发现在无氮液体培养基中保藏6年的Frankia.菌丝断裂,孢囊不完整.同期经有氮液体保藏的Frankia菌孢囊较完整.冷冻干燥保藏3.5年和砂管保藏8年,孢囊和菌丝均较完整.上述方法保藏的菌种,经活化后均能生长,且具有典型的Frankia菌形态特征和固氮活性.4种方法比较,无氮液体保藏法的菌体细胞生长速度快,固氮活性强,有侵染结瘤能力.     

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.     

Static magnetic field selects undifferentiated myelomonocytes from low-glutamine concentration stimulated U937 cells

Reduced glutamine (GLN) concentration in the culture medium of a U937 cell line caused them to be differentiated along the monocytic pathway; cells attached to the matrix and to each other by extending pseudopodia and acquired specific functional characteristics, such as the expression of alpha-naphthyl-acetate esterase and the capacity to reduce nitroblue tetrazolium, as well as becoming active phagocytes. When U937 cells were differentiated under continuous exposure to a 6mT static magnetic field (MF) the overall differentiation process was perturbed. Surprisingly, after 5 days' exposure to the static MF, higher cell viability and differentiation were observed in cells cultured in a GLN-deprived medium than in cells grown in the same medium but in the absence of a static MF. The latter cells, particularly those that were still floating in the medium, were stimulated with TPA for a further 3 days. These cells differentiated and attached to the substrate. Conversely, the same treatment applied to cells cultured in GLN-deprived medium in the presence of the static MF resulted in resistance to TPA-induced differentiation. Indeed, these cells exhibited a round shape and in-suspension growth.     

Evaluation of N-acetylchitooligosaccharides as the main carbon sources for the growth of intestinal bacteria

N-Acetylchitooligosaccharides ((GlcNAc)(n)) with different degrees of polymerization (n=1-6) were prepared as the main carbon sources in media for evaluating the growth of nine intestinal bacteria. A chitohydrolysate was prepared by hydrolyzing shrimp-shell chitin using HCl. After purification, the purity of each (GlcNAc)(1-6) was >86%. The growth of intestinal bacteria was carried out in a basal medium (BM) containing 0.2% (w/v) of each sugar or glucose as the main carbon source and was evaluated using maximum cell densities and specific growth rates. Bacteroides fragilis and Clostridium perfringens could respectively utilize GlcNAc and (GlcNAc)(2) more efficiently for growth than glucose. Bifidobacterium adolescentis and Eubacterium limosum could use (GlcNAc)(1-6) slightly as their main carbon source. Escherichia coli, Lactococcus lactis and Proteus vulgaris could utilize glucose more efficiently than (GlcNac)(1-6). GlcNAc was used more readily than (GlcNAc)(2-6) by Staphylococcus aureus, exhibiting almost the same specific growth rates. In BM, Streptococcus faecalis grew well even without adding each of the sugars tested.     

Isolation and growth characteristics of nitrilotriacetate-degrading bacteria

Two bacterial strains that degrade nitrilotriacetate (NTA) were isolated from NTA-acclimatized activated sludge. These bacteria grew well in NTA medium with optimal pH around 7. The growth rate constants of the bacteria, strains N-2 and N-5, were 0.046 h⁻¹ and 0.11 h⁻¹ at the concentration of 0.1% NTA (pH 7.0, 25°C), respectively.The growth of each bacterium was inhibited at high concentrations NTA. The growth rate decreased roughly linearly with increasing concentration of NTA. The strains N-2 and N-5 showed maximal cell growth at the concentrations of 0.2% and 0.25% NTA, respectively. The strain N-2 would not grow at the concentration of 0.5% NTA. On the other hand, the strain N-5 showed a little growth under the same conditions. Also, the bacterial growth was almost completely inhibited when divalent metal ions such as Mg⁺⁺, Ca⁺⁺, and Fe⁺⁺ were omitted from the culture medium, or slightly excess EDTA (1 mM) was added to the medium. These results suggest that the bacterial growth inhibition at high concentration of NTA is caused by the sequestration of metal ions in the medium.     

Growth and sporulation of Beauveria bassiana and Metarrhizium anisopliae on media containing various amino acids

Natural isolates of two entomogenous fungi, Beauveria bassiana and Metarrhizium anisopliae, were cultured in liquid culture media containing 24 amino acids and KNO₃ to determine their effect on growth and sporulation. In addition, the growth and medium pH changes for each isolate grown on an asparagine-containing medium were compared. Tryptophan and alanine were most effective for growth and sporulation of B. bassiana, although glutamine and KNO₃ also produced large numbers of regularly shaped spores. Tryptophan, glutamic acid, and histidine were all well utilized for both growth and sporulation of M. anisopliae. Nitrogen sources containing sulfur were poorly utilized for sporulation by M. anisopliae. In general, B. bassiana produces greater mycelial mass and much larger numbers of spores than M. anisopliae. Both fungi attained nearly the same growth maximum on asparagine medium though B. bassiana exhibited an initially more rapid growth rate. In both fungi this rapid growth phase was accompanied by a decline in medium pH followed by a rise in pH during the decline phase of growth.     

Serum free fed batch production of IgM

Summary Fed batch cultivation of a murine hybridoma secreting IgM in a serum free medium was successfully attempted. Cell growth and IgM productivity remained high for over a month, and compared well to a similar fed batch culture in medium supplemented with 10% fetal calf serum. The average specific secretion rates of antibody in both media were the same. Sufficient inoculum cell density was crucial to the establishment of a viable culture in serum free medium for the cell line, NS6.3, used.     

上海地区藓类环境生理学特性的初步研究

对22种上海地区藓类植物进行了包括抗旱性、温湿度及光照、生长基质及组织培养等环境生理学方面的研究．结果表明,在同样环境条件下不同种的抗旱表现不同,生长在不同生境下的同一种的表现亦不同;沙壤添加有机质和短光照有利于藓类的生长;适当的激素处理可有效地促进藓类植物的繁殖生长;Hogland培养基适用于藓类植物的组织培养     

The in vitro propagation of amaryllis (Hippeastrum spp. hybrids)

A new, rapid technique for the propagation of amaryllis (Hippeastrum spp. hybrids) by means of tissue culture is reported. Leaf bases, scapes, peduncles, inner bulb scales and ovaries were cultured successfully in vitro and plantlets were induced readily at various concentrations of growth regulators. Some plantlets also were produced in the absence of growth regulators. The most productive tissues for propagation were inverted scapes and peduncles, cultured in a modified Murashige and Skoog salt solution with added organic constituents and 1 mg per 1 (4.5 micron) 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg per 1 (4.4 micrometer) 6-benzylaminopurine (BAP). Plantlets induced axenically also grew roots on the generalized shoot-inducing medium so that no special rooting medium was required. Although friable callus was obtained from ovary tissue cultured on a medium containing 2 mg per 1 (11 micrometer) naphthaleneacetic acid and 4 mg per 1 (18 micrometer) BAP, it produced shoots after 8 weeks of further subculture on the same medium. An average of 10 rooted plantlets was obtained from each scape or peduncle explant on the shoot-propagating medium. Thus, if 45 explants are obtained from each bulb, 450 rudimentary plantlets could be obtained from each mother bulb in 8 weeks of culture. This is a substantial increase over present propagation methods.     

巴尔通体液体培养条件简化及生长曲线观察

【目的】应用一种昆虫细胞培养基作为基础成分培养巴尔通体(Bartonella species),建立一种操作方便、高效稳定的巴尔通体液体培养方法。【方法】昆虫细胞培养基中添加10%胎牛血清,以此为基础培养液分别添加蔗糖和谷氨酰胺,比较这两种成分对汉赛巴尔通体(B.henselae)和五日热巴尔通体(B.quintana)生长的影响并观察其他10种巴尔通体在简化后的培养液中的生长特性。【结果】添加蔗糖和谷氨酰胺不会明显促进巴尔通体的生长,10种巴尔通体在简化后的培养液中均生长良好。不同种巴尔通体生长曲线不同,汉赛巴尔通体和五日热巴尔通体的世代时间分别为5.2 h和4.3 h,生长速度快于固体培养。【结论】以昆虫细胞培养基作为基础成分的培养液适于巴尔通体液体培养,特别是对一些更难培养的巴尔通体提供了一种较好的培养方法。     

The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

Secondary somatic embryogenesis and plant regeneration in myrtle (Myrtus communis L.)

Immature seeds, as well as hypocotyls and cotyledons excised from seedlings of Myrtus communis L., were cultured on media containing half-strength Murashige and Skoog macronutrients (MS/2) with combinations of auxins and cytokinins, in order to study their morphogenetic competence. Somatic embryogenesis was obtained from cotyledons, hypocotyls and 2-month-old immature seeds with 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The percentage of explants showing this primary somatic embryogenesis ranged from 4% for hypocotyls to 12% for 2-month-old immature seeds. In the latter, somatic embryogenesis was also obtained in media containing 2,4-D plus a cytokinin, and with only a cytokinin. Somatic embryos obtained from hypocotyls, cotyledons or immature seeds were able to develop on MS/2 medium without plant growth regulators. Subculture of primary somatic embryos obtained from immature seeds on MS/2 medium without plant growth regulators gave rise to clusters with secondary somatic embryos and embryogenic calli. These clusters were subcultured every 8 weeks, and they were the source of highly embryogenic cultures. An average of 10% of the secondary somatic embryos developed into plantlets in each subculture. Therefore, the same culture on MS/2 medium without growth regulators yielded both plantlets and de novo secondary embryos. Received: 6 April 1998 / Revision received: 10 July 1998 / Accepted: 21 July 1998     

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.     

Factors influencingin vitro micropropagation ofPinus strobus L.

Anin vitro procedure for micropropagation ofPinus strobus L., consisting of the following four steps has been established: shoot induction, shoot growth, root induction, and root growth. Influence of certain selected factors on each of these steps was determined. Shoot induction was found to be influenced by the age of the explant as well as the concentration of 6-benzyladenine in the medium. Best shoot growth was obtained on the medium of Litvayet al. without any growth regulators. Addition of activated charcoal to shoot growth medium resulted in fewer shoots. Root induction on several media was compared. Best root induction occurred when shoots were put on a half strength Gresshoff and Doy’s medium supplemented with 0.5 to 1.0 mg r1⁻¹ α-naphthylacetic acid (NAA) for two weeks. Shorter exposure of shoots to higher concentrations of NAA or indol-3-ylbutyric acid (IBA) was not as effective. Low temperature seemed to be a requirement for root growth.     

The location of the arginine-specific carboxypeptidase in the membrane of Mycoplasma salivarium and its physiological functions

A non-penetrating probe, 2,4,6-trinitrobenzenesulfonate, inhibited the activity of the carboxypeptidase purified from the cell membranes of Mycoplasma salivarium and the same enzymatic activity of intact Mycoplasma cells as well. Growth of the organism in medium containing benzoylglycyl-L-arginine resulted in a higher pH and higher turbidity than growth in the same medium without this supplement. It was concluded that the enzyme existed in the outer surface of the membrane of the cells and probably functioned to supply the organism with arginine as an energy source.     

Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica

Hairy root clones of Scopolia japonica were established by selection of adventitious roots formed on the root segments inoculated with Agrobacterium rhizogenes strain 15834. Twenty-nine isolated hairy root clones displayed various phenotypes characterized by growth rate, opine production and tropane alkaloid production. Of these, two highly alkaloid productive clones SI and S22 were examined for their growth rate and alkaloid productivity under various cultural conditions. When the most scopolamine-productive clone SI was cultured for 4 weeks at 25°C in the dark, the weight of the root tissue was increased by 40 times and the content of scopolamine reached a level of 0.5% on a dry weight basis in each optimum medium. On culture of the most hyoscyamine-productive clone S22 under the same conditions as with S1, the weight was increased by 102 times and the content of hyoscyamine was 1.3% on a dry weight basis in each optimum medium.     

Establishment and characteristics of Gottingen minipig skin in organ culture and monolayer cell culture: relevance to drug safety testing

Skin from Gottingen minipigs was used as a source of tissue for organ and cell culture and compared to human skin for growth conditions and sensitivity to irritants. Optimal organ culture conditions were determined, based on the preservation of the histological structure. These included serum-free, growth factor-free conditions with a calcium concentration of 1.5mM. Formulations in which the calcium concentration were low (0.075-0.15mM) failed to support tissue viability (even in the presence of dialyzed serum). Epidermal keratinocytes were grown from tissue explants and as single cells from enzyme-disrupted tissue. Optimal keratinocyte growth was achieved using a serum-free, growth factor-supplemented culture medium with a calcium concentration of 0.15mM. Fibroblasts were optimally grown from explant cultures using a medium with 1.5mM calcium and 10% fetal bovine serum. The conditions that were optimal for maintenance of intact pig skin, as well as for the isolated cells, are the same conditions that have been shown previously to be optimal for intact human skin and skin cells. In additional studies, pig skin keratinocytes and fibroblasts were exposed to a panel of contact irritants and contact sensitizers. Using growth inhibition as the response, the median effective dose values with each agent were very similar to the values previously determined for human epidermal keratinocytes and human dermal fibroblasts. Taken together, these data suggest that the skin from the Gottingen minipig can be used as a surrogate for human skin in ex vivo skin safety studies.