| CHO cells (Chinese hamster ovary cell) is presently the most important expression systems for engineering antibodies and recombinant protein and serum-free fed-batch culture is also the most important process for biopharmaceuticals production. Deeply Understanding CHO cells metabolic dynamics would be favor of improving serum-free fed-batch CHO cells culture technology. Accordingly, the main purpose of this study is to establish the dynamic model of cell growth and metabolism and build the approximate index intermittent fed-batch strategy in order to provide the reference theory for achieving optimal control of serum-free fed-batch culture. The global convergence optimal parameters of cell growth, substrate consumption and feed frequency were estimated by non-linear programming through using the global convergence Levenberg-Marquardt method based on MATLAB software. At the same time, according to the infiltration and promotion impact of genomics research on cell culture technology development, the gene transcription characteristics of the cells cultured in serum-free fed-batch mode was investigated by using gene chip technology in order to understand the underlying reasons about the growth and metabolic characteristics of the cells cultured in serum-free fed-batch mode and provide clues for exploring the possible way to promote cells in more effective growth and metabolism state.Establishment of a serum-free fed-batch CHO cells culture paltform which can reflect a certain extent about the characteristics and technical indexes of fed-batch culture process was the basis and premise for this research. The recombinant CHO cells expressing recombinant human pro-urokinase (Pro-UK) as the object of study. And both the cells cluster and injury in suspension culture due to the shear stress were effectively eliminated by adding Pluronic F-68 and dextran sulfate. Compared with the original cells, the average growth rate of the cells adapted to suspension culture in logarithmic phase increases from 0.27-1 to 0.48-1, and the highest viable cell density, increased from 2.5×106 cells/ml to 6.3×106 cells/ml. It was shown that the cells adapted to suspension culture showed similar metabolism characteristics to the original cells.An appropriate serum-free medium for CHO cell suspension culture was formulated by application of Plackett-Burman and response surface method. The maximum cells density reached 4.2×106 cells/ml in serum-free suspension batch culture mode and was about 1.7 fold of that in 1% serum-containing suspension batch culture, correspondingly the maximum activity of interest increased about 1.6 fold.A serum-free fed-batch CHO cells culture platform was established through investigating the impact of the start time of feeding, the initial cell inoculation density, the different culture components and substrate limits strategy on fed-batch culture. The results showed that the maximum cells density reached 7.8×106 cells/ml and the largest protein activity reached 8875 IU/ml in fed-batch culture during 12 d culture process, which were about 85.7% and 50.9% increase as compared with those of the batch culture, respectively.In consideration of the fact that a whole fed-batch culture process is actually compose of both a batch culture stage and a feeding culture stage, The growth and metabolic characteristics of the cells in both serum-free batch culture stage and serum-free fed-batch culture stage was investigated individually. According to the established mathematical model of cell growth, substrate consumption and product formation, the optimal mathematical model parameters of logarithmic cell growth phase was estimated by non-linear programming using the global convergence of the Levenberg-Marquardt method and the MATLAB software based on the actual experimental data. The dynamic models of cell growth and metabolism in serum-free batch culture, which could match the experimental results and reflected essentially inherent orderliness about cell growth, glucose consumption and lactic acid production of the cells in logarithmic growth phase stage were established Further, according to the established mathematical model of cell growth, substrate consumption and feed frequency, the optimal mathematical model parameters of the cells in feeding culture stage were estimated by non-linear programming using the global convergence of the estimated Levenberg-Marquardt method and the MATLAB software again, based on the actual experimental data. Thus, the dynamic models of growth and metabolism of the cells in fed-batch serum-free culture and the approximate index for intermittent feeding strategy were established, which were approved to be feasibility in guiding serum-free fed-batch culture control in a 2 L bioreactor.The differences of gene expression levels of the cells in different culture phase in both batch and fed-batch modes were revealed by using gene chip technology. And based on the known metabolic pathway or the cell signaling pathway, the expression level differences of genes related to glucose metabolism, apoptosis and cell cycle of the cells cultured in batch and fed-batch modes were analysed by using Genmapp software. Results from this study showed that among approximate 19,191 the target gene in gene chip, the number of down-regulated genes was higher than that of up-regulated genes of the cells in both batch and fed-batch culture. Further, the number of down-regulated genes of the cells in fed-batch culture was much higher than that of the cells in the recession phase of batch culture. The results suggested that more glucose was utilized through glycolysis pathway by the cells in batch culture according to the key regulatory genes expression of glycolysis and TCA cycle in batch culture and fed-batch culture process. And, due to down-regulated of the key genes regulated glycolysis in the fed-batch culture stage, its would potentially bring about reducing the ratio of glucose incomplete oxidation and increasing the ratio of glucose of complete oxidation of glycolytic. There was no significant difference in the gene expression, which were related to the key apoptosis pathways of the cells cultured in batch and fed-batch mode, only a few gene expression changed, which illuminated the results of cell apoptosis proportion by using flow cytometry. According to the key regulation gene expression of cell cycle in both culture mode, the results indicated that the cell proliferation and cells viability were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, cyclin and CKI family and up-regulating Smad4 genes, which also explained clearly the differences of cell cycle distribution in culture mode. Overall, in two culture modes, the cells achieved controlling cell metabolism, proliferation and the cycle and responded to changed culture conditions through down-regulating gene expression with reference to cell metabolic pathway or signaling pathway.The above results show that metabolism dynamics model of serum-free fed-batch cells culture based on the MATLAB software and the global convergence Levenberg-Marquardt method reflected the actual cell growth and culture volume change of fed-batch cell culture process and provided a theoretical reference basis for realizing optimal control in serum-free fed-batch CHO cells culture. At the same time, the study about gene transcription characteristics of the CHO cells cultured in serum-free fed-batch culture will not only help to clarify the underlying causes of cell growth and metabolism differences between the CHO cells cultured in batch mode and fed-batch mode as well as to provide clues for exploring possible ways to promote cells in a more effective growth and metabolism state.
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