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Primary cell lines are invaluable for exploring cancer biology and investigating novel treatments. Despite their numerous advantages, primary cultures are laborious to obtain and maintain in culture. Hence, established cell lines are still more common. This study aimed to evaluate a range of techniques for isolating primary breast cancer cultures, employing distinct enzymatic compositions, incubation durations, and mechanical approaches, including filtration. Out of several protocols, we opted for a highly effective method (Method 5) that gave rise to a primary cell culture (BC160). This method combines mechanical disaggregation and enzymatic digestion with hyaluronidase and collagenase. Moreover, the paper addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations. To make primary cell lines a preferred model, it is essential to elaborate and categorise isolation methods, develop approaches to separate heterogeneous cultures and investigate factors influencing the establishment of primary cell lines., (© 2023. The Author(s).)

Cancer-associated fibroblasts are a highly heterogeneous group of cells whose phenotypes and gene alterations are still under deep investigation. As a part of tumor microenvironment, they are the focus of a growing number of studies. Cancer-associated fibroblasts might become a new target of breast cancer therapy, but still more tests and analyses are needed to understand mechanisms and interactions between them and breast cancer cells. The study aimed to isolate cancer associated fibroblasts from breast cancer tissue and to phenotype the isolated cell lines. We focused on various cancer-associated fibroblast characteristic biomarkers and those that might differentiate various cancer-associated fibroblasts' subtypes. Patients with a histological diagnosis of invasive breast cancer (diameter ≤15 mm) and qualified for primary surgical treatment were enrolled in the study. Cell lines were isolated from breast cancer biopsy. For the phenotyping, we used flow cytometry, immunofluorescence and RT-qPCR analysis. Based on our study, there was no indication of a clear pattern in the cancer-associated fibroblasts' classification. Results of cancer-associated fibroblasts expression were highly heterogeneous, and specific subtypes were not defined. Moreover, comparing cancer-associated fibroblasts divided into groups based on BC subtypes from which they were isolated also did not allow to notice of any clear pattern of expressions. In the future, a higher number of analyzed cancer-associated fibroblast cell lines should be investigated to find expression schemes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (AJCR Copyright © 2022.)