Development of a serum-free medium using computer-assisted factorial design and analysis

Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA Analysis of variance - BSA bovine serum albumin - CHO Chinese hamster ovary - FBS fetal bovine serum - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - PBS phosphate buffered saline - SFM serum-free medium     

Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells

Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular -galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular -galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.     

The crystal violet nuclei staining technique leads to anomalous results in monitoring mammalian cell cultures

Nuclear counts determined by crystal violet staining from samples of stationary or microcarrier cultures of hybridomas, CHO or Vero cells were consistently and significantly higher than cell concentrations determined by the trypan blue or Coulter counter methods. This difference was attributed to the presence of a significant proportion of binucleated cells, which are assumed to be 35% of the cell population in the stationary phase of Vero cultures. The proportion of such cells during exponential growth was variable. However, continuous sub-culture of these cells induced a degree of synchrony during growth which resulted in a cyclic variation of the difference between the cell and nuclei counting techniques. This data indicates that care should be taken in interpreting cell culture profiles based solely on crystal violet nuclei staining counts.     

大鼠卵巢绒毛膜促性腺激素受体在CHO中的表达和扩增

本文报道了利用二氢叶酸还原酶放大系统将大鼠ＬＨ／ｈＣＧ受体（记为Ｆ－ｈＣＧＲ）及其胞外肽段（记为Ｔ－ｈＣＧＲ）在中国苍鼠卵巢细胞（ＣＨＯ）中的表达。ＳＤＳ－ＰＡＧＥ分析表明,Ｆ－ｈＣＧＲ为一条蛋白质带,其表观分子量为９２ｋｄ,而Ｔ－ｈＣＧＲ为３５ｋｄ和３７ｋｄ两条带。表达受体对其配基ｈＣＧ表现出高的亲合力,Ｆ－ｈＣＧＲ的解离常数为７×１０－９ｍｏｌ／Ｌ,Ｔ－ｈＣＧＲ为６．４×１０－９ｍｏｌ／Ｌ。表达Ｆ－ｈＣＧＲ的转染ＣＨＯ细胞可结合１２５Ｉ－ｈＣＧ,而表达Ｔ－ｈＣＧＢ者不结合１２５Ｉ－ｈＣＧ。这提示Ｆ－ｈＣＧＲ主要存在于细胞质膜表面上。表达Ｆ－ｈＣＧＲ的转染ＣＨＯ细胞能刺激。ｃＡＭＰ的形成,而表达Ｔ－ｈＣＧＲ者不能刺激ｃＡＭＰ形成。免疫荧光定位结果表明,Ｔ－ｈＣＧＲ主要分布于质膜的细胞质侧以及胞内其他一些细胞器膜上。用免疫亲和层析可以得到纯化的Ｔ－ｈＣＧＲ。     

t-PA cDNA在CHO细胞中的高效稳定表达

我们曾报道t-PA mRNA非翻译区序列对其表达有明显的抑制作用,在此基础上,通过对5′-UTR及3′-UTR的改造,使t-PA在COS-7细胞中的表达水平提高30倍左右。将t-PA表达质粒用电击法转染中国仓鼠卵巢细胞二氢叶酸还原酶缺陷株(CHO-dhfr),经过混合加压及筛选,在CHO细胞中高效表达了t-PA,表达水平达到5000～6000 IU/10~6细胞/24hr。重组t-PA具有与天然t-PA相同的分子量及酶活性。经过8个月连续传代,表达水平未下降,表明细胞株是稳定的,其主要指标均符合工程细胞株的要求。     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

Observations on the influence of glutamine,asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.     

Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction ( 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.