Isolation of label-free and viable circulating tumour cells (CTCs) from blood samples of cancer patients through a two-step process: negative selection-type immunomagnetic beads and spheroid cell culture-based cell isolation

Isolation of high-purity, label-free, and viable circulating tumour cells (CTCs) from cancer patients is crucial for subsequent analyses. To address this issue, a two-step CTC isolation scheme was proposed, wherein a spheroid cell culture was used to further purify viable CTCs after conventional negative selection-based CTC isolation methods. Our results from a cancer cell line model revealed that the survival of leukocytes in spheroid cell cultures was significantly decreased with time, whereas OECM-1 cells maintained viability and proliferated. Therefore, such a cell culture operation was expected to increase cancer cell purity in the cell spheroids. This assumption was confirmed by our results, which showed that cancer cell purities were 10.6 to 80.3-fold increased after spheroid cell culture for 8 days. In the following clinical tests, CTC-related cells were observed in 6 of 13 blood samples. Furthermore, the average purity of CTC-related cells was 34.8 ± 14.0%. By utilizing a second-step spheroid cell culture operation, the purity of CTC-related cells was greatly improved when compared with that (less than 10%) achievable by conventional negative selection-based CTC isolation. Overall, this study proposed a two-step process for the isolation of high-purity, label-free, and viable CTCs.