The performance of a glass bead shaking technique for the disruption of Escherichia coli cells

The efficacy of a simple laboratory method for cell disruption based on the shaking of glass beads on a rotary shaker was assessed in this study, via measurements of the release of total protein and interferon-α2b from E. coli. The optimum conditions for cell disruption were detected after 30 min of shaking in Tris-HCl buffer (pH 8) at 300 rpm with 1.5 g of glass beads (diameter: 0.5 mm) per mL of cell suspension volume. Three test runs were conducted under the above conditions and the maximum average protein release values were determined as 3.048, 3.564, and 3.015 mg/mL, respectively. The amount of protein release was comparable to the amount of protein release in ultrasonica-tion and glass bead vortexing procedures. The amount of interferon-α2b release in the ultrasonication, glass bead vortexing, and glass bead shaking trials were 240, 172, and 201 ng/mL, respectively. This method was shown to process between 1 and 10 mL of sample volume in a 50 mL Falcon tube without a great deal of deviation, and was able to handle in excess of 60 samples simultaneously.     

ZtNH2-HCl和剪切力对茶叶细胞悬浮培养中茶氨酸合成的影响

以婺源绿茶嫩叶愈伤组织为材料,在采用摇床悬浮培养与发酵罐悬浮培养．分析了茶氨酸合成前体盐酸乙胺(ZtNH2-HCl)不同的添加方式和剪切力对培养细胞增长量和茶氨酸合成量的影响。结果显示,发酵罐放大培养取得了与摇床悬浮培养类似的效果;在一个培养周期中,培养细胞茶氨酸积累高峰出现在第20～22天;发酵罐大规模培养时采用桨叶式搅拌器(低剪切力)细胞增长量和茶氨酸合成苗优于标准板搅拌器;添加盐酸乙胺可大幅度提高茶氨酸积累量,先加入一定量盐酸乙胺再每天进行少量补充,茶氨酸合成最比一次性加入的效果要好。     

樟叶越桔细胞悬浮培养条件的优化

为了提高樟叶越桔(Vacciniumdunalianum)悬浮培养细胞的生物量,以樟叶越桔叶片愈伤组织为试材,通过单因素试验探究不同蔗糖浓度、培养基pH值、培养基体积、初始接种量和摇床转速对悬浮培养细胞生长的影响,并根据响应面法Box-Behnken试验设计原理进行组合试验以优化培养条件。结果显示,以改良WPM培养基为基础培养基,樟叶越桔细胞悬浮培养的最优条件为40 g·L^–1蔗糖、培养基pH5.2、培养基体积45 mL、初始接种量2.64 g和摇床转速为149 r·min^–1,其细胞生物量干重为0.184 4 g,与理论预测值0.184 5 g较为接近,且细胞的生长曲线呈S型。研究结果为樟叶越桔悬浮培养细胞次生代谢产物的生产调控奠定了技术基础。     

An extracellular macromolecular complex from the surface of soybean suspension cultures

A cell-free, insoluble cell wall fraction is described which floats on the surface of suspension cultures of Glycine max L. Merrill var. Acme cells. Its accumulation is governed by both the shaking speed and the medium volume, a shaking speed of 110 to 120 revolutions per minute with a medium volume of about 100 to 120 milliliters in a 250-milliliter flask being optimal. Various factors which could control the accumulation of the complex were tested and are discussed, and scanning electron micrographs of the complex being released from the cell surface are presented.     

油菜游离细胞培养及易再生的体细胞无性系的建立

以芥菜型油菜(Brcssica juncea)下胚轴来源的愈伤组织为材料,进行悬浮细胞振荡培养,得到游离的单细胞。这些细胞作浅层液体静置培养,可获得高频率的愈伤组织。在附加有水解乳蛋白200毫克/升、BA 2—3毫克/升及GA,0.1—1毫克/升的MS培养基上,这些愈伤组织分化了芽。分化的芽在无激素或附加有0.01—0.1毫克/升IAA或NAA的MS培养基上长出根,从而发育成完整植株。当对游离细胞获得的再生植株的茎切段愈伤组织,再经上述程序进行细胞培养时,发现愈伤组织诱导率及愈伤组织分化频率均远远高于原供体,从而建立了B.juncea易再生的体细胞无性系。     

A rapid method for determination of acid phosphatase activity of whole yeast cells

A simple and rapid procedure for determination of intracellular acid phosphatase activity without the need for disruption of cells is described. Candida lipolytica cell suspension was treated with 0.1% Triton X-100 for 30 min at room temperature and with intermittent shaking. The enzyme assay is carried out directly with the permeabilized cell suspension. Permeabilization of the yeast cells to p -nitrophenylphosphate by Triton X-100 provides almost 100% efficiency in determining the total acid phosphatase activity compared to results obtained with disrupted yeast cells.     

The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins

Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.     

Cyanide production from glycine by a homogenate from a Pseudomonas species.

A cell-free preparation with cyanide-producing activity was obtained from a bacterium, strain C, of the genus Pseudomonas. To preserve activity, an oxidizing agent, e.g., phenazine methosulphage (PMS), had to be added to the cell suspension before disruption by sonic treatment. By the procedure described, a total homogenate made from a 15% (wet weight) bacterial suspension in tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.05 M, pH 8.2) and with PMS (0.4mM) exhibited about 8% of the activity obtained from a suspension of untreated bacteria. In the presence of flavine-adenine dinucleotide (0.3 mM) and PMS (0.4mM), the activity was augmented to about 16% of that of the intact cells. By gradient centrifugation the homogenate was separated into three fractions. The main enzyme activity was associated with those fractions which by electron microscopy were found to consist of membranous structures.     

Efficient formation of cell spheroids using polymer nanofibers

Spheroid culture has been used for suspension cultures of anchorage-dependent cells. In this study, we developed a new method for the suspension cultures of anchorage-dependent animal cells using polymer nanofibers. Poly(lactic-co-glycolic acid) nanofibers (785?nm in average fiber-diameter, 88?μm in average fiber-length) fabricated by the electrospinning method were added to each suspension culture of human embryonic kidney 293 cells and human dermal fibroblasts. As compared to no addition of nanofibers to the suspension cultures, nanofibers enhanced cell spheroid formation, thereby reducing cell death resulting from a lack of cell adhesion. Efficient formation of spheroids in the presence of polymer nanofibers may be useful for the suspension cultures of anchorage-dependent cells.     

Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies

Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested industrially in pilot scale with conventional linear membrane microfiltration modules, their performances were severely limited due to membrane fouling. The purpose of this work was to determine whether Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) could be used to improve filtration performance over that observed with conventional linear cross-flow microfiltration (CMF). For the microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspension as feed, solute transport (as measured by mass) was from 100 to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. In the presence of the neutral surfactant, Tween 20, solute transport for diafiltration was at least 25 times higher during the first 10 min of operation and 100% higher after 300 min with the helical module as compared with that obtained with the linear module. Clearly, improved filtration performance, a purer and more concentrated product, and substantial savings can be expected with the new Dean vortex filters.     

Establishment of a human somatic hybrid cell line for recombinant protein production

Cell fusion techniques were used to derive mammalian host cell lines suitable for large-scale production of therapeutic proteins. Although the 293S cell line, of human embryonic kidney origin, is an excellent host cell for mammalian gene expression, these cells have a tendency to form large and tight aggregates in suspension cultures and bioreactors. To solve the problem of aggregation, 293S cells were fused to a human suspension cell line, 2B8 (a Burkitt's lymphoma derivative), using polyethylene glycol (PEG). The PEG-treated 293S and 2B8 cells were selected in a medium supplemented with hypoxanthine-aminopterin-thymidine and G418 (1 mg/ml) to eliminate nonfused cells. These hybrid clones, designated as HKB (hybrid of kidney and B cells), are negative for endogenous immunoglobulin expression. Most clones are readily adaptable to serum-free suspension culture under shaking conditions without forming large and tight aggregates. One clone, HKB11, was shown to support high-level expression of cytokines [interleukin (IL)-2 and IL-4], ICAM-1 and rFVIII in a side-by-side comparison with 293 and Chinese hamster ovary cells. The above-described characteristics of HKB cells indicate that HKB11 is a favorable cell host for the production of human therapeutic proteins.     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.     

Defense mechanisms of mice against Fonsecaea pedrosoi infection

Defense mechanisms of a host against Fonsecaea pedrosoi infection were studied histopathologically using athymic nude (nu/nu) mice of BALB/c background and their heterozygous (nu/+) littermates. Thirty male nu/nu and 30 nu/+ mice, weighing 16–19 g, were employed in this experiment. The nu/nu or nu/+ mice were divided into 3 groups consisting of 10 each. Furthermore, 4 nu/nu mice were supplemented to investigate effects of lymph node cell transfer. Subglobose cells of F. pedrosoi Tsuchiya strain were obtained from a culture in brain heart infusion glucose (1%) broth with reciprocal shaking at 37 °C for 17 days, and then 0.02, 0.1 and 0.5% cells suspensions were prepared. Each cell suspension was allotted to one group of the nu/nu or nu/+ mice. 0.1 ml of the cell suspension was inoculated into a tail vein, then one mouse from each group was sacrificed 1, 2, 4, 6, 8, 10, 14, 18, 21 and 25 days after inoculation. In both the nu/nu and nu/+ mice, the brain, kidneys and heart were affected severely with the strain in that order. Histopathologically, the defense mechanisms of the nu/+ mice against the fungus infection consisted chiefly of 2 steps: first, of non-immune phagocytosis by polymorphonuclear leucocytes (PMNs), and second, of granuloma formation induced by cell-mediated immunity. Those of the nu/nu mice consisted only of one step: phagocytosis by PMNs. A difference in susceptibility to the strain between the nu/nu and nu/+ mice changed according to the amount of the fungal cells inoculated. When inoculated with the 0.02% cell suspension, the resistance of the nu/nu mice was stronger than that of the nu/+ mice. In contrast, when inoculated with the 0.5% cell suspension, the former was affected more severely than the latter. There were little differences in the susceptibility to the strain between the nu/nu and nu/+ mice inoculated with the 0.1% cell suspension. These data seem to indicate that the phagocytic function of PMNs of the nu/nu mice was more active than that of the nu/+ mice, and the nu/nu mice inoculated with the 0.5% cells suspension (beyond the phagocytic capacity) lost resistance against the fungus infection. When the nu/nu mice were transferred with lymph node cells before inoculation of the strain, granulomata were formed to prevent hyphae from growing freely in the tissue.     

Uptake of dehydroepiandrosterone-3-sulfate by isolated trophoblasts from human term placenta, JEG-3, BeWo, Jar, BHK cells, and BHK cells transfected with human sterylsulfatase-cDNA

The human placenta lacks the enzyme 17-hydroxylase/17-20-lyase, and is thus unable to convert cholesterol into estrogens. Therefore estrogen synthesis of trophoblast cells depends on the supply of precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16-hydroxy-dehydroepiandrosterone-3-sulfate by maternal and fetal blood. To investigate the cellular internalisation of these anionic hydrophilic precursors, the uptake of [³H]-/[³⁵S]-DHEA-S and [³H]-taurocholate by isolated cytotrophoblasts, cells of choriocarcinoma cell lines (JEG-3, BeWo, Jar), BHK and BHK cells transfected with human sterylsulfatase-cDNA (BHK-STS cells) was studied. Furthermore, the activity of sterylsulfatase of these cells in suspension and in corresponding cell homogenate was measured.

During the first 5 min of incubation with [³H]-DHEA-S or [³⁵S]-DHEA-S, radioactivity of cytotrophoblasts increased significantly, while radioactivity of JEG-3, Jar, BHK and BHK-STS cells did not increase. Radioactivity of BeWo cells increased slightly. For all cell types, there was no significant difference for uptake of either substrate. During incubation with [³H]-taurocholate, radioactivity of cytotrophoblasts did not increase. Sterylsulfatase activity of cytotrophoblast homogenate was significantly lower than that of cytotrophoblast suspension. Sterylsulfatase activity of BHK-STS, JEG-3 or BeWo cell homogenate was significantly higher than that of the corresponding cell suspension. In BHK and Jar cells sterylsulfatase activity was not detectable.

Cytotrophoblasts take up DHEA-S without prior hydrolysis. BHK, BHK-STS, JEG-3, and Jar cells do not take up and BeWo cells slowly take up DHEA-S. In cytotrophoblasts extracellular DHEA-S rapidly gains access to intracellular sterylsulfatase, while in choriocarcinoma and BHK-STS cells access of DHEA-S to sterylsulfatase is limited. Our results indicate, that uptake by cytotrophoblasts is mediated by a carrier which is not expressed in choriocarcinoma or BHK cells and which is different from the known taurocholate-transporting organic anion transporting polypetides.     

A method of shaking-blotting--a simple and reliable means for obtaining direct chromosomal preparations from chorionic biopsies

The method proposed is based on a gradual fixation, and short-term hydration before softening and on a new technique of chromosome preparation. The latter is based on the sample softening in 60% acetic acid directly on the slide, its gentle shaking and spot-blotting, a careful spreading of the cell suspension on slide surface avoiding the usage of micropipets and other handlings causing metaphase plate breakage. The method provides a highly efficient karyotyping of human embryos within the 7-12th weeks of gestation.     

In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

An efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10⁸ cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.     

Thymidine incorporation of bacteria sequentially extracted from soil using repeated homogenization-centrifugation

Bacteria were sequentially extracted from soil into a water suspension after shaking soil with water or mixing it in a blender followed by a low-speed centrifugation. Bacteria, which were released only after several cycles of homogenization-centrifugation, had higher growth rates as judged from thymidine and leucine incorporation, whereas bacteria that were more readily released by a gentle shaking procedure had the lowest growth rate. This indicated that bacteria more tightly bound to soil particles were growing faster than those that were more easily released into the water suspension. The same pattern was found both in an agricultural and a forest soil, with contrasting pH and organic matter content, and irrespective of whether the bacteria were labeled before or after the centrifugation steps. The different growth rates of the bacteria could not be explained by different partitioning of label between different macromolecules, different cell size, different viability of the bacteria, or different dilution of the added radioactive substrate in the different homogenization-centrifugation fractions. The total amount of phospholipid fatty acids per bacterial cell was also similar in the different fractions. Different composition of the bacterial communities in the different homogenization-centrifugation fractions was indicated by a gradually altered phospholipid fatty acid pattern of the extracted bacteria, and an increased hydrophobicity of the bacteria released only after several homogenization-centrifugation treatments. Correspondence to: E. Bååth     

Metabolism of 4-hydroxynonenal by the rat hepatoma cell line MH1C1

The metabolism of the toxic lipid peroxidation product 4-hydroxynonenal was investigated in the well-differentiated rat heptoma cell line MH1C1. When exposed to 0.1 mM 4-hydroxynonenal (HNE), MH1C1 cells consumed it in a time-dependent manner. There was a linear relationship between the amount of aldehyde consumed and cell number in the range 0.5 - 4 X 10(6) cells ml-1. This process was unaffected by pyrazole, suggesting that alcohol dehydrogenase is not involved. The whole homogenate of MH1C1 cells consumed added HNE at a rate similar to that in intact cells. Fractionation of the homogenate showed that the highest HNE-metabolizing activity is in the cytosol. The dialysed cytosol had almost no capacity to metabolize HNE, but this was restored by supplementation with NAD, NADH, NADP and NADPH. The metabolism of HNE in MH1C1 cells is thus different from that in hepatocytes, which were shown to utilize cytosolic alcohol dehydrogenase for this process. Both reductive and oxidative pathways could be implicated in the metabolic activity of MH1C1 cells towards HNE as well as binding by glutathione.     

A new photometric method for oxygen consumption measurements in cell suspensions

A new technique is described for measuring O2 consumption rates and O2 concentrations in suspensions of respiring cells. Aliquots of a cell suspension kept in a special thermostated precision syringe are injected into the measuring system in defined time intervals. The O2 content of these samples is determined photometrically, as reported previously. The O2 consumption per cellular wet weight and/or per single cell can be calculated from the cell volume fraction, the physical density, the cell concentration in the suspension, and the time-dependent decline of the O2 concentration in the precision syringe. The minimum detectable amount of O2 is 0.1 microliter O2, which corresponds to 0.001 (vol/vol) of O2 if a 100-microliters sample of suspended cells is analyzed. Reproducibility of the O2 consumption measurement is 9% of the measured value. The advantages offered by this method are the straightforward calibration in absolute terms, the short time required for one analysis (2-6 min), a high sensitivity, the simultaneous determination of overall O2 concentration and O2 consumption rates in cell suspensions, and the great variability in the application.