This study evaluates methods for isolating primary breast cancer cell cultures, focusing on enzymatic digestion, mechanical disruption, and filtration. Five protocols were tested, with Method 5 being the most effective, yielding a primary cell line (BC160) through a combination of mechanical and enzymatic digestion using hyaluronidase and collagenase. The study addresses challenges in isolating primary cultures, particularly the competition between fibroblasts and cancer cells. Primary cell cultures are valuable for studying cancer biology and personalized therapies, but they are difficult to isolate and maintain. The research highlights the importance of optimizing isolation methods to obtain pure, viable cell cultures. The study also discusses the heterogeneity of primary cultures, including the presence of cancer-associated fibroblasts, and the impact of cell composition on culture outcomes. The results show that Method 5 provides a stable primary cell line, while other methods were less effective or more complex. The study emphasizes the need for standardized protocols and further research to improve the isolation and maintenance of primary cell cultures. The findings suggest that enzymatic digestion combined with mechanical methods is more effective than purely mechanical or enzymatic approaches. The study also highlights the importance of cell culture medium optimization for successful primary cell line establishment. The results demonstrate that BC160, a primary breast cancer cell line, exhibits characteristics similar to established cell lines, including gene expression patterns and cell surface markers. The study concludes that primary cell lines are essential for cancer research and personalized therapy, but their isolation and maintenance require careful optimization of methods and conditions.This study evaluates methods for isolating primary breast cancer cell cultures, focusing on enzymatic digestion, mechanical disruption, and filtration. Five protocols were tested, with Method 5 being the most effective, yielding a primary cell line (BC160) through a combination of mechanical and enzymatic digestion using hyaluronidase and collagenase. The study addresses challenges in isolating primary cultures, particularly the competition between fibroblasts and cancer cells. Primary cell cultures are valuable for studying cancer biology and personalized therapies, but they are difficult to isolate and maintain. The research highlights the importance of optimizing isolation methods to obtain pure, viable cell cultures. The study also discusses the heterogeneity of primary cultures, including the presence of cancer-associated fibroblasts, and the impact of cell composition on culture outcomes. The results show that Method 5 provides a stable primary cell line, while other methods were less effective or more complex. The study emphasizes the need for standardized protocols and further research to improve the isolation and maintenance of primary cell cultures. The findings suggest that enzymatic digestion combined with mechanical methods is more effective than purely mechanical or enzymatic approaches. The study also highlights the importance of cell culture medium optimization for successful primary cell line establishment. The results demonstrate that BC160, a primary breast cancer cell line, exhibits characteristics similar to established cell lines, including gene expression patterns and cell surface markers. The study concludes that primary cell lines are essential for cancer research and personalized therapy, but their isolation and maintenance require careful optimization of methods and conditions.