3.1. Time Course Transcriptome Heterogeneity in Suspension Cultures
We performed single-cell Quartz-Seq as previously reported (Sasagawa et al., 2013) of 85 D4, 70 D8, and 78 D11 CHO cells. To compare the transcriptomes of the cells cultured for different lengths of time (days), we performed PCA of all the expression data (Figure 1). The proportion of variance in principal component 1 (PC1) was 0.3102, while that of PC2 was 0.077. Using this approach, we could quantitatively evaluate the cellular heterogeneity, which previously was not possible. The standard deviations in PC1 were 1634 for D4, 3024 for D8, and 5029 for D11. We rejected the null hypothesis that heterogeneity did not increase during culture, based on the Bartlett test for the homogeneity of variances in the PC1 between different culture times (p < 2.2e-16). Thus, the cellular heterogeneity increased during cultivation and this increase showed no sign of attenuation. Hence, the maximum attainable heterogeneity of the CHO cells in a long-term high cell density culture was not predictable from these data. To assess cellular heterogeneity in future bioengineering processes, very long-term cultures such as perfusion continuous cultures need to be studied. We can control it if it can be measured.