Development of a practical small-scale circulation bioreactor and application to a drug metabolism simulator

We developed an easy-to-use, small-scale circulation-type bioreactor system that enables the simultaneous evaluation of many specimens. Medium flow was generated by a magnetic stirrer in this system. Primary rat hepatocytes formed a monolayer, and there were no morphological differences between cells in circulation and stationary cultures. The mitochondrial activity of hepatocytes in the circulation culture was 23% lower than that in the stationary culture after 2 days of culture. On the other hand, albumin production activity in the circulation culture after 2 days of culture was 1.4 times higher than that in the stationary culture. Albumin production activity per cell in the circulation culture was 1.9 times higher than that in the stationary culture after 2 days of culture. In addition, lidocaine metabolism rate per cell in the circulation culture was 1.3 times higher than that in the stationary culture. The lidocaine clearance of the circulation culture in our circulation-type bioreactor was 1.3 times higher than that of the stationary culture. It was shown that this bioreactor is suitable for the expression of the liver-specific functions of primary rat hepatocytes. Therefore, we can expect that this circulation-type bioreactor system will be a practical drug metabolism simulator.     

甜(辣)椒单倍体培养研究进展

介绍了甜(辣)椒花药培养和游离小孢子培养的研究概况,花药培养应用相对成熟,游离小孢子培养尚未获得突破性进展。对影响花药培养的各关键因素(包括材料基因型、供体植株生长状态、小孢子发育时期、培养基、培养方法、变温处理、培养条件等)进行了综述,并讨论了甜(辣)椒单倍体培养存在的问题和进一步研究方向。     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

手工和仪器两种血培养结果差异性分析

目的对手工法双相血培养瓶和BACTEC9120全自动血培养仪的阳性率作回顾性分析。方法将血液标本同时接种双相血培养基和BACTEC9120全自动血培养仪配套血瓶中,将阳性结果移种血平板,如为阴性再移种巧克力平板。结果370例血培养,双相血培养瓶阳性25例,阳性率为6．76％（25／370）,树脂需氧（儿童）瓶BACTEC9120报警显示阳性59例,阳性率为15．9％（59／370）,阳性标本移种到血平板及巧克力平板阳性54例,阳性率为14．6％（54／370）,假阳性5例,假阳性率为1．4％（5／370）,共有29例树脂需氧（儿童）瓶阳性,而双相血培养瓶为阴性,P〈0．001。结论BACTEC9120全自动血培养仪提高阳性率,缩短阳性的报告时间优于传统的双相血培养基。     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

抗感枯萎病西瓜根际微生物比较研究

本文通过传统微生物培养方法,研究了在土培和基质培条件下,抗感枯萎病西瓜不同生育期根际和非根际微生物数量的变化.结果表明,微生物数量在根际显著高于在非根际微生物,且随西瓜生长发育的阶段不同而变化,苗期根际微生物数量最少,以后随西瓜生长发育,根际微生物数量不断增加,至生长旺盛的开花结果期,微生物数量达到最高,在西瓜生长发育后期,根际微生物数量又有所回落.西瓜抗枯萎病性与根际细菌的数量具有相关性,在生长发育各个阶段,无论是土培还是基质培,均表现为抗病材料的根际细菌数量高于感病材料的根际细菌数量.根际真菌与放线菌数量与西瓜的抗感枯萎病性没有相关性.非根际微生物数量在整个生育期变化辐度较小.非根际细菌数量在土培条件下几乎保持在同一水平,在基质培条件下迅速增加,至生长后期有所回落.非根际真菌与放线菌数量在土培和基质培条件下均于生长后期达到最高.     

不同培养方法对甘薯无菌苗生长的影响

试验结果表明,在甘薯组织培养中液体培养不降低无菌苗生长势,对无菌苗生长有一定促进作用,适合于甘薯无菌苗的快速繁殖。液体培养20d比相同条件下固体培养的无菌苗植株高度高出63．16％,经方差分析,差异显著,具有统计学意义;液体培养60d比相同条件下固体培养的无菌苗植株高度,单株叶片数,单叶面积分别高出59．43％,106．70％,49．82％,经方差分析,前两者差异显著,具有统计学意义。     

Damaging agitation intensities increase DNA synthesis rate and alter cell-cycle phase distributions of CHO cells

The effects of fluid-mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2-L bioreactors has been examined. A two-color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high-agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S-phase cell of the high-agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high-agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250-rpm culture had a higher fraction of S-phase and a lower fraction of G1-phase cells than the 235-rpm culture. The DNA sunthesis rate per viable S-phase cell of the 250-rpm culture was greater than of the 235-rpm culture. (c) 1992 John Wiley & Sons, Inc.     

大麦不同基因型游离小孢子的直接培养再生及培养体系的优化

以微搅拌法建立了小孢子直接游离的预处理和培养程序。在大田生长的４个对培养反应不同的大麦基因型上,以新鲜幼穗游离小孢子进行直接培养,均成功地诱导了胚状体并获得再生绿色植株。小孢子的发育进程说明,直接游离的小泡子在预处理过程中的发育要慢于在花药中预处理的小孢子,而且其培养效率也较低。直接游离小孢子的培养密度以０．８～１．０×１０５／ｍｌ较理想,至少应不低于６×１０４／ｍｌ．８％－１０％的糖浓度可明显提高小孢子分裂频率和胚状体诱导频率。实验结果也表明两种培养基ＦＨＧ和ＭＮ６无明显差异,均适宜于直接游离的小孢子培养,并对游离小孢子直接培养在理论和应用上的意义进行了讨论     

培养和冷冻对新生大鼠胰岛活性的影响

新生大鼠胰碎片在MEM、DMEM、M-199和RPMI-1640中培养,比较培养不同天数冷冻保存,不同条件处理及液氮贮存不同时间的实验结果。表明新生大鼠胰碎片在RPMI-1640中培养胰岛素分泌量最高,随着培养时间的延长,胰岛素分泌量逐渐升高。培养6d进行冷冻保存为宜。新生大鼠胰碎片在液氮中贮存12个月分泌功能无明显变化。     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Zymomonas mobilis

The production of ethanol from starch by a coimmobilized mixed culture system of aerobic and anaerobic microorganisms in Ca-alginate gel beads was investigated. The mold Aspergillus awamori was used as an aerobic amylolytic microorganism and an anaerobic bacterium, Zymomonas mobilis, as an ethanol producer. By controlling the mixing ratio of the microorganisms in the inoculum size, a desirable coimmobilized mixed culture system, in which the aerobic mycelia grew on and near the oxygen-rich surface of the gel beads while the anaerobic bacterial cells mainly grew in the oxygen-deficient central part of the gel beads, was naturally established under the aerobic culture conditions, and ethanol could be directly produced from starch by the system. The ethanol productivity by the system in flask culture was particularly affected by the shear stress (dependent on the shaking speed) which controlled the mycelial growth on the surface of the gel beads. Under optimum culture conditions in the flask culture, the glucose produced was instantly consumed, and was not observed in the culture broth; the final concentration of ethanol produced from 100 g/L starch was 25 g/L and the yield coefficient for ethanol, Y(pls), was 0.38. The ethanol productivity by the coimmobilized mixed culture system was compared with those by other various culture systems and the advantages of the system were clarified.     

Studies on Cell Suspension Culture and Fermentation Culture in Arnebia euchroma

This paper reports some characteristics of cell suspension and fermentation culture in Arnebia euchroma (Royle) Johnst. The yield of suspension culture reached 22.0g dry wt/L per month when inoculum quantity was 2.50 g dry wt/L. Time-course study showed that cell growith lagged in 0–3 days and enhanced greatly in 3–12 days, and almost ceased after 12 days of culture, pH value changed during the culture period and peaked on the 12th day after inoculation. When cells were cultured in liquid production medium, the contents of shikonin derivatives increased quickly and reached to the maximum about the 25th day. The cell yield of 9.47 and 9.34 g dry wt/L per month was obtained in fermentation culture. Timecourse of cell growth in fermentation culture was similar to that in suspension culture. The total content of shikonin derivatives in fermentation culture was 14.26% dry weight from 10 L bioreactor. The yield of shikonin derivatives was 1.93 g/L.     

Setting up a Single Cell Clone Lines of Cathamus tinctorius

Different concentrations of 2,4-D, KT and NAA were able to influence the plating efficiency (PE) of single cells of Cathamus tinctorius. The best combination of these three hormones for the growth of single cells was 2.0, 0.3 and 0.5 mg/l, respectively. The PE was obviously different as cells came from different generations of suspension subculture and the third generation of suspension culture cells, had the best PE which 8.5 times as high as that of the first generation of suspension culture cells. Single cell growth in condition medium or in solid-liquid dual layer culture was better than in normal plate culture. The PE of single cell clones in condition culture was 3.6 times as high as in normal plate culture. The PE of single cell clones in solid-liquid dual layer culture was 4.7 times as high as in normal plate culture. Many clones from single cells were set up. Different growth rates were observed in different single-cell clones. The lowest growth rate in these clones was 3.08 g/g/35 days, the highest growth rate in these clones was 23.33 g/g/35 days.     

红花单细胞克隆的建立

KT,2,4-D 及 NAA 能提高红花(Cathamus tinctorius)细胞克隆平板培养的植板率。这三种激素对细胞生长的最佳搭配是2,4-D2.0mg/l,KT0.3mg/l,NAA 0.5mg/1。红花细胞悬浮继代培养代数不同,其植板率相差甚远,用悬浮培养第三代的细胞做材料最好,其植板率是第一代悬浮培养细胞做材料的8.5倍。红花细胞克隆的条件培养的植板率是普通平板培养的3.6倍。固-液双层培养的植板率是普通平板培养的4.7倍。对已建立的红花细胞克隆进行生长速率的比较表明,生长最漫的克隆的生长速率为3.08g/g/35天,生长最决的克隆的生长速率高达23.33g/g/35天。     

Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal

An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture. Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l(-1) was added to the culture during the late exponential-growth phase. The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion. The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture. In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1+/-7.5 mg l(-1), which was about seven times the amount obtained in the non-elicited batch culture. The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l(-1) day(-1), which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture.     

Conditions of the primary culture for rat hepatocytes and plasminogen activator release

The conditions of primary culture for rat hepatocytes was investigated on the releasing effect of Plasminogen Activator (PA). The culture method using Collagen Coated Dish (CCD-method) which is currently available and the ordinary culture method using Plastic Culture Dish (PCD-method) were employed for that purpose in a comparative way. The effect of the addition of some supplements, that is FN, Aprotinin, EGF were also investigated. The following results were obtained. The dissociated rat hepatocytes formed a monolayer with pavementlike morphology at 24-48 hours after seeding. No difference was observed in the morphology of hepatocytes during the culture period between the two methods, although CCD-method allowed 120 hours culture, whereas PCD-method allowed 72 hours. The PA activity was demonstrated on the hepatocytes by either culture method according to the fibrinolysis autography. The cultured hepatocytes released PA into the medium continuously as long as the viability and morphology of the cells were maintained in good state. The PA activity reached the maximum after 96 hours culture in CCD-method, whereas it reached the maximum after 48 hours in PCD-method. The addition of Aprotinin to the culture medium was not necessary for PA release in CCD-method in contrast to PCD-method. When EGF was discontinued in the culture medium, the release of PA was reduced in association with the occurring of morphological disintegration of hepatocytes.     

Vero细胞微载体不同培养方法的评价

对三种不同培养方法进行细胞生长速度、密度、营养及代谢产物浓度的比较分析,以优化和筛选最佳培养条件与方式。用同体积生物反应罐,基本培养条件相同,采用批培养、再循环培养、灌流培养三种方式进行了Vero细胞微载体（CytodexI）的周期培养。三种培养方法均达到预期效果,最终细胞密度分别为每毫升2.09×10     

牛肠激酶催化亚基工程菌培养条件的探索与鉴定

目的:探索牛肠激酶催化亚基工程菌高效表达的培养条件。方法:牛肠激酶催化亚基基因的初步表达,筛选构建工程菌,从以下方面入手优化培养条件:培养基的组成、摇瓶发酵培养条件、培养基的PH、种龄、接种量、培养时间等,并且通过Western Blotting和SDS-PAGE等鉴定不同条件下的实验结果,从而确定最佳培养条件。结果:通过鉴定实验结果,从不同种平行培养条件中选择产量最高的培养条件,作为最优培养条件。结论:成功地探索并鉴定了工程菌的适宜培养条件。     

垄作与覆膜对川中丘陵春玉米根系分布及产量的影响

通过设置田间试验,研究种植方式(垄作垄播、平作、垄作沟播)与覆膜措施对川中丘陵春玉米根系分布和玉米产量的影响.结果表明: 垄作与覆膜对玉米根系形态影响显著,覆膜显著提高了玉米根长、根表面积和根体积,拔节期覆膜较不覆膜处理分别提高了42.3%、50.0%和57.4%.覆膜提高了各土层和各水平各层次根质量,扩大了根系在土壤垂直和水平方向上的分布,提高了拔节期20～40 cm土层和水平方向上宽行0～20 cm根系比例.种植方式对根系生长和分布的影响因覆膜而异,覆膜下垄作垄播显著提高了各土层根质量和20～40 cm土层根量分配比例,同时提高了水平方向上各层次根量和宽行根系分配比例,总根质量表现为:垄作垄播>平作>垄作沟播;不覆膜下垄作沟播显著提高了窄行0～40 cm根量,吐丝期总根质量表现为:垄作沟播>垄作垄播>平作.从玉米穗部性状和产量看,覆膜降低了玉米秃尖长,提高了穗长、穗粒数、千粒重和产量,覆膜下产量表现为:垄作垄播＞平作＞垄作沟播;不覆膜下则表现为:垄作沟播＞平作＞垄作垄播.覆膜下垄作垄播促进了根系特别是深层根质量的增加,同时增加了深层根系和水平20～40 cm根系比例,这是其增产的重要原因;而不覆膜下垄作沟播有利于根系生长,从而提高产量.     

Culture conditions of Aspergillus oryzae for production of enzyme preparation

Submerged culture was better than solid culture in the production of proteinase and peptidases from Aspergillus oryzae 460. On the contrary, solid culture was better than submerged culture in the production of α-amylase, carboxymethyl cellulase, and pectinlyase from the same fungus.The soy souce mash (moromi) made with the enzyme preparation from submerged culture was highly viscous and the soy sauce produced was characteristic in low contents of alcohol and reducing sugar, low pH value, and less aroma. Soy sauce made with the enzyme preparation from solid culture was superior on these points to that from submerged culture.Wheat bran was best as the raw material for the enzyme preparation in easy koji making, large amount produced, and low cost.In enzyme production from a solid culture, addition of urea (0.8% to wheat bran) nearly doubled the leucine aminopeptidase for Leu-Gly-Gly. The incubation period was reduced to 30 to 40 h from 50 to 60 h using germinated spores and moisture-controlled culture with forced aeration.