Investigation and Enhancement of Growth Characteristics of Chinese Hamster Ovary Cells

Background.

Since Chinese hamster ovary (CHO) cells were isolated from Cricetulus griseus in 1957 by Theodore T. Puck (Tjio and Puck, 1958), they have been extensively used for research purposes and for the production of recombinant therapeutic proteins. The advantages of CHO cell lines compared to their microbial counterparts are correct folding of complex proteins, a high secretion rate and human-like post translational modifications (Wurm, 2004).
The major drawback is the overall productivity, since space-time yields are ten to hundredfold lower than in prokaryotic hosts (Krampe and Al-Rubeai, 2010). This depends mostly on the growth rate, the specific productivity, the stress resistance and the stability (Müller et al., 2008). One possibility to increase space-time yield is to increase the initial growth rate of CHO cells so as to faster reach high cell densities for efficient production.

Aims and methods.

The aim of this project is to investigate and manipulate factors which influence the growth rate of CHO K1 cells. This will be achieved by 3 different approaches:

• As serum containing CHO cells are faster growing than cells which are adapted to serum-free conditions (Zanghi et al., 1999), it was decided to identify the differentially regulated mRNAs, microRNAs (miRNAs) and signalling pathways by microarray analysis. Candidates showing high correlation with growth rate will then be overexpressed or knocked down in serum-free cell lines to investigate the effect on these.





• Certain gene and miRNA candidates, which are annotated in the literature to enhance the growth rate, were chosen for overexpression to check the beneficial potential in the CHO K1 cell line. After cloning and transient transfection of these, the most promising candidates will be chosen to create stable clones for further investigations.


• In a method called dye dilution, cells are stained with a fluorescence dye, which is lost according to the specific growth rate of the cell. It has been observed that subpopulations with different growth rates arise from a uniformly stained starter culture. These subpopulations will be separated by fluorescence analysed cell sorting (FACS) and the different mRNA and miRNA expression patterns will be analysed by microarrays to find out why these subpopulations emerge. The results will be used to find additional candidates which can improve the growth characteristics.
  

Krampe, B. and M. Al-Rubeai (2010). Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies. Cytotechnology 62(3): 175-188.
Müller, D., H. Katinger, et al. (2008). MicroRNAs as targets for engineering of CHO cell factories. Trends Biotechnol 26(7): 359-365.
Tjio, J. H. and Puck, T. T.(1958).Genetics of somatic mammalian cells. II. Chromosomal constitution of cells in tissue culture. J Exp Med 108(2): 259-268.
Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotech 22(11): 1393-1398.