This study evaluates various techniques for isolating primary breast cancer cultures, focusing on enzymatic compositions, incubation durations, and mechanical approaches. Out of several protocols, Method 5, which combines mechanical disaggregation with enzymatic digestion using hyaluronidase and collagenase, was found to be highly effective, leading to the establishment of a stable primary cell line (BC160). The method involves a prolonged incubation period, ensuring cell viability and homogeneity. The study also addresses common issues in isolating primary cultures, such as the struggle against fibroblasts overgrowing cancer cell populations. The importance of optimizing isolation methods and developing approaches to separate heterogeneous cultures is emphasized, highlighting the need for further research to improve the reliability and reproducibility of primary cell line isolation. The successful isolation of a stable primary BC cell line (BC160) with characteristic gene expression patterns suggests the potential of primary cell lines in personalized cancer therapy.This study evaluates various techniques for isolating primary breast cancer cultures, focusing on enzymatic compositions, incubation durations, and mechanical approaches. Out of several protocols, Method 5, which combines mechanical disaggregation with enzymatic digestion using hyaluronidase and collagenase, was found to be highly effective, leading to the establishment of a stable primary cell line (BC160). The method involves a prolonged incubation period, ensuring cell viability and homogeneity. The study also addresses common issues in isolating primary cultures, such as the struggle against fibroblasts overgrowing cancer cell populations. The importance of optimizing isolation methods and developing approaches to separate heterogeneous cultures is emphasized, highlighting the need for further research to improve the reliability and reproducibility of primary cell line isolation. The successful isolation of a stable primary BC cell line (BC160) with characteristic gene expression patterns suggests the potential of primary cell lines in personalized cancer therapy.