Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Repeated-batch cultures of Baby Hamster Kidney cell aggregates in stirred vessels

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate chacteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this selection procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.     

Development of media for the mixotrophic/heterotrophic culture ofBrachiomonas submarina

The marine algaBrachiomonas submarina var.pulsifera (Droop) CCAP 7/2A, is employed as a food organism in aquaculture; it can be cultured heterotrophically or mixotrophically. Growth rates and productivity under heterotrophic conditions were lower than those achieved under mixotrophic conditions. By reducing the osmotic potential of the medium, whilst simultaneously increasing the levels of nitrogen and phosphorus and using sodium acetate as a carbon source, a 20-fold increase in final yield was attained. This corresponded to a maximum culture of 9.02times 10⁶ cells ml^–1 and a dry weight of 2.51 g l^–1.Author for correspondence     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

沙打旺组织培养中脱分化细胞的超微结构及细胞核的动态变化

对沙打旺的下胚轴、子叶、幼叶组织培养中脱分化细胞进行了超微结构观察,并着重讨论了细胞核的动态变化。脱分化细胞的细胞质中线粒体墙加,嵴明显;多聚核糖体增多;高尔基体增加;质体中积累淀粉。核仁与核内异染色质之间有一个动态过程。此过程暂称“核仁物质喷射“现象。在致有以下：１．核体出现,半嵌在增大的核仁上,核内异染色质沿核膜凝聚;２．异染色质移向核仁,并与核仁接触,核体消失,部分异质进入核仁;３．核仁物质     

蛇床幼茎离体培养中体细胞胚胎形成的观察

蛇床幼茎外植体经诱导产生了愈伤组织。在ＭＳ＋２,４－Ｄ,０．２ｍｇ／Ｌ＋ＺＴ０．４ｍｇ／Ｌ培养基中,愈伤组织转变成胚性愈伤组织。转入ＭＳ＋ＮＡＡ０．２ｍｇ／Ｌ＋ＺＴ０．８ｍｇ／Ｌ培养基以后,胚性愈伤组织分化出体细胞胚胎。体细胞胚胎在ＭＳ＋ＮＡＡ０．５ｍｇ／Ｌ培养基中可直接发育成为完整植析。显微观察表明,体细胞胚胎产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有螺纹导管的分化。子叶期的维管组织从两     

玉米胚性愈伤组织的长期继代及其染色体分析

对５种基因型幼胚诱导的愈伤组织继代培养表明,玉米胚性愈伤组织的长期继代受基因型,培养基成分,激素,培养条件的影响。适时继代,逐代筛选对胚性保持起重要作用。适当降低培养温度（１２±２℃）有利于愈伤组织的保存和胚性保持,可以减少愈伤组织长期继代所需的物质和工作量。长期继代培养的胚性愈伤组织,胚状体发生能力和植株再生率无显著变化,但正常苗的再生频率显著下降。观察愈伤组织细胞染色体发现：（１）基因型对不同倍性细胞的比例有明显影响。（２）随着继代时间的延长,二倍体细胞下降,四倍体和非二倍体细胞增多。（３）愈伤组织中出现多种染色体结构变异,这些结构变异有可能导致非整倍体细胞的形成。     

葡萄试管苗热处理结合茎尖培养脱病毒效果的研究

对７个生食葡萄品种的试管苗热处理结合茎尖剥离培养脱除病毒的方法做了研究,结果表明,从热处理试管苗剥离的茎尖培养成活率为６１．２％,其中有１８．１％的成苗。各品种热处理试管苗剥离的茎尖分化再生能力不同：先形成愈伤组织的成苗率较低,而先分化芽的茎尖成苗率较高。这一方法对扇叶病毒和卷叶病毒的脱除率达９２％以上。