Figure 1. Summary of novel methodology for establishment of OL0825 CTC line.

Subcutaneous implantation of 1×10⁶ BNL 1ME A.7R.1 cells into the flank of a Balb/c mouse resulted in tumor development in the flank. Humane euthanization of mouse involved CO₂ asphyxiation and intracardiac exsanguination. 500–1000 µl of whole blood was obtained. After centrifugation, buffy coat layer was subjected to red blood cell lysis, washed in PBS and cultured in medium in a humidified incubator at 37°C with air and 5% CO₂. Tumor cell proliferation was observed within 48 hours. The cell line was designated OL0825 and is still very viable after 25 passages and repeated freezing and thawing. Phase contrast image of OL0825 was taken at ×40 magnification (objective lens).