Serum-free cell growth and differentiation studies in cultured cell lines

The thesis is divided into two main sections investigating growth and differentiation in cultured cell lines. Investigations were carried out into the establishment of serum-free media (SFM) for the long-term subcultivation of the industrially important cell line, CHO-K1. When the cell line was cultured in a high calcium containing medium, cellular growth was found to be inhibited after 4 to 6 passages on average. However, by growing the cells in a low calcium medium, continuous cultivation was possible. It appears that the calcium concentration in the SFM is critical to this long-term growth. The SFM developed were also capable of supporting growth of CHO-K1 in suspension culture with Pluronic F68 supplementation and in microcarrier culture. Serum-free media was also developed for the growth and sub-cultivation of a human lung cancer cell line, DLKP. This cell line contains three morphologically distinct subpopulations. The growth pattern of the three clones differed in serum-free medium with only one of them, DLKP-I, capable of proliferating in the above medium. However, the addition of fibronectin to the SFM permitted growth of the other two clones. All sub-populations could be subcultured indefinitely in SFM. DLKP-A, a multiple drug resistant variant of the parental DLKP cell line, was also successfully grown and subcultured in SFM. Differentiation studies were also carried out on DLKP. This cell line was originally histologically diagnosed as a ‘poorly differentiated carcinoma of the lung’. Treatment of the cells with the differentiation-inducing agent, 5-bromodeoxyuridine (BrdU) resulted in the induction of integrin and upregulation of a2p, integrin expression. Associated with this change in integrin expression was a functional change in the properties of the cells themselves with BrdU treatment. The cells developed different adhesive properties showing more rapid attachment to the extracellular matrix proteins, collagen, laminin and fibronectin, and to basement membrane compared to untreated cells. Addition of an anti-integrin blocking antibody to BrdU-treated cells reversed the changes in adhesive properties of the cells. BrdU treatment of DLKP cells also induced the expression of a number of epithelial antigens, including epithelial specific antigen (a cellular adhesion molecule) and keratin 19 filaments. A significant upregulation of keratin 19 protein expression was also observed in the human lung adenocarcinoma cell line, A549, after BrdU treatment. Preliminary RT-PCR analysis has shown that the effect of BrdU on keratin 19 mRNA expression appears to be at the post-transcriptional/ translational level