Oxygen transfer in animal cell culture medium

Both k(L)a and k(L) measurements were carried out by an unsteady state technique at impeller speeds ranging from 1.6 to 5.8 s(-1) in a mechanically agitated animal cell culture vessel of working volume 1.5 L. Checks were made that the time constant of the oxygen electrode was negligible compared to the time for aeration and that the oxygen electrode reading was not a function of agitator speed in the range employed. The k(L) values by surface aeration of (1.18-3.54) x 10(-5) m/s and k(L)a values by sparged aeration of (2.8-8.5) x 10(-4) s(-1) were found. The former are in reasonable agreement with published experimental values and the latter in accord with values estimated from published correlations based on agitator power input and aeration rate. The fluids used were water, basal medium, and basal medium supplemented with 5% (v/v) foetal calf serum; for each of these, k(L) and k(L)a values were similar. However, the addition of silicone antifoam (6 PPM) reduced the k(L)a value by ca. 50%.     

Effects of microcarrier concentration in animal cell culture

Results are presented which show how the microcarrier concentration affects the hydrodynamic environment in animal cell bioreactors. At low levels of agitation, no physical effects of microcarrier concentration were found. However, cell growth was strongly influenced by cell concentration. At high levels of agitation, a strong detrimental effect of microcarrier concentration was found. A new mechanism of hydrodynamic damage was identified which is second order in microcarrier concentration. The identification of this mechanism adds to the fundamental understanding of hydrodynamic phenomena in microcarrier bioreactors.     

Simulation and dynamic optimisation of animal cell culture

In animal cell culture, there are some 25 substrates that both have a significant effect on the culture performance and which can be measured with relative ease. A detailed dynamic simulation for such a culture has been produced and an optimisation policy that use this model to identify ideal media conditions has been developed. This paper describes an extension of that work to include the dynamic optimisation of cultures under fed-batch operation. Two different types of feeding policy were considered – in the first, discrete shots of feed were supplied, while in the second, feed was added continuously. Both policies offered significant improvements in the predicted productivity of the culture - up to 30% that of an experimentally optimisedbatch culture.     

A cell culture model of chemically and spontaneously derived mouse lung alveologenic carcinoma

Malignant cell lines related to mouse lung alveologenic carcinoma have been established from urethane-induced tumors and after in vitro spontaneous transformation of preneoplastic cell lines. Both the chemically and spontaneously transformed cell lines formed invasive, poorly differentiated carcinomas with secondary lung deposits when implanted subcutaneously in immune-suppressed mice. They differed from the related preneoplastic cell line in coordinately exhibiting anchorage-independent growth, reduced epidermal growth factor receptor activity and absence of pericellular fibronectin. These data suggest that similar molecular events may occur in type 2 pneumocyte-related cells in order to generate mouse lung alveologenic adenomas and carcinomas by both spontaneous and chemical carcinogen induction mechanisms. A reduced level of pericellular fibronectin was also demonstrated in an in situ compressive urethane-induced mouse lung adenoma. Loss of pericellular fibronectin may therefore be an early and persistent phenotypic alteration during transformation to the alveologenic adenoma and carcinoma.Abbreviations EGF epidermal growth factor - TGF transforming growth factor     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

Functional and evolutionary analyses on expressed intronless genes in the mouse genome

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life--bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.     

Applications of alkaline protease fromConidiobolus in animal cell culture

Summary Alkaline protease fromConidiobolus has been tested as a substitute for trypsin in animal cell culture. Applications in the dissociation of cells for primary cell cultures, maintenance of cell lines and the production of G-bands on metaphase chromosomes are described. The advantages of a microbial enzyme for such applications are discussed.     

Viscous reduction of turbulent damage in animal cell culture

Animal cells are exposed to turbulent fluid flow in many cell culture processes. If the turbulence in the flow is sufficiently strong, the cells will be damaged or killed by fluid-mechanical forces. Through an increase in viscosity, the turbulence can be damped and the hydro-dynamic damage can be reduced. In this article, new experimental results are presented which illustrate the protective effect of thickening agents. The results follow the prediction of a model based on Kolmogorov's theory of universal equilibrium in turbulent flow fields.     

Methods for integration site distribution analyses in animal cell genomes

The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host–virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences.     

Characteristics of CHO-K1 cell culture producing two types of recombinant soluble thrombomodulin

Recombinant CHO-K1 cells, expressing human soluble thrombomodulin, were cultured in a serum-free medium and characteristics of the culture associated with glucose and lactate were investigated. In 3 L fermentor (3LFM) cultures, the cell density was found to have a proportional relationship with the volumetric glucose consumption rate, and the specific glucose consumption rates were constant at about 0.2 mg/(10⁶ cells·d) despite many differences in the culture conditions. Thus, it was concluded that the glucose consumption rate is little influenced by the condition of the cells or the culture conditions, and that the cell density can be estimated by the glucose consumption rate calculated from glucose measurement. Two types of thrombomodulin (rsTMα and rsTMβ) were produced, in which rsTMβ possesses chondroitin-4-sulfate and has greater anticoagulant activities than rsTMα. Therefore, it is important to investigate the rsTMα and rsTMβ production properties, and to determine the optimal culture conditions for high rsTMβ production. The most important factor to increase the production of rsTMβ relative to rsTMα (the β/α ratio) was effective aeration. Moreover, a lower ratio of lactate production/glucose consumption (the L/G ratio) with sufficient oxygen, high glucose concentration, and a longer medium exchange interval contributed to a higher specific rsTMβ production rate. Since there was a linear relationship between the production rate of each type of rsTM and the overall rsTM production rate per liter, it is expected that the rsTMα and rsTMβ production rates may be able to be estimated from the overall rate and the rsTMβ production increased by increasing the overall rsTM production with a lower L/G ratio.     

Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal-embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.     

Colorimetric pH measurement of animal cell culture media

Most animal cell culture media can be buffered using bicarbonate and high pressure CO₂ in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO₂ pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.