Corneal endothelial cells maintained in tissue culture retain the ability to synthesize and secrete an extracellular matrix (ECM) along their basal cell surface. Treatment of confluent cultures with 0.5% Triton X-100 results in the removal of the cell monolayer, thereby exposing the ECM, which adheres strongly to the tissue culture dish. Dishes coated with ECM were used to study the permissive effect of such a substrate on cell proliferation. The proliferation of bovine granulosa and adrenal cortex cells maintained on plastic tissue culture dishes was compared to that on dishes coated with ECM. Neither cell type, even when exposed to optimal serum concentration, replicated when seeded at low cell density on plastic. In contrast, when seeded on ECM they proliferated actively. None of the cultures maintained on ECM required fibroblast growth factor in order to reach confluence, although when maintained on plastic they were totally dependent on fibroblast growth factor for proliferation. Because cells maintained on plastic do not respond to factors present in serum or plasma, although they do so respond when maintained on ECM, it is likely that the close contact of the cells with the ECM restores their sensitivity to agents present in serum and plasma.

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A major issue in the use of mammalian cell culture in biopharmaceutical manufacturing is the removal of process related impurities, such as residual host cell DNA, during the product purification process. To ensure that sufficient DNA removal is achieved during purification, it is essential to have an accurate and sensitive assay for host cell DNA. The quantitative polymerase chain reaction (QPCR) is widely used for this purpose; however, the extent to which the choice of QPCR gene target can have an impact on final results requires further understanding. In the present study, we examined the relationship between the genomic copy number of eight different Chinese Hamster ovary (CHO) gene targets and the sensitivity and accuracy afforded by those targets in a residual host cell DNA QPCR assay. We also evaluated the use of each gene target for accurate measurement of residual DNA clearance using in-process purification samples from two CHO production cell lines. Our results revealed a correlation between gene target abundance and the potential sensitivity for use in a QPCR assay. However, we found that higher copy number gene targets do not provide the highest measurement or reveal the largest clearance of residual host cell DNA from purification samples. These findings suggest that different DNA sequences may clear or degrade at differential rates and highlight unexpected considerations that must be made in the choice of QPCR gene target when designing QPCR assays.

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