Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma that contributes to tumor progression, therapeutic resistance, and disease aggressiveness. Cancer-associated fibroblasts (CAFs) represent the predominant cellular component of this stroma and are a major source of extracellular matrix (ECM), playing a central role in shaping the tumor microenvironment. However, the reliable isolation of CAFs from human PDAC tissue remains technically challenging due to extensive matrix deposition, limited tissue availability, and an elevated risk of microbial contamination. In this study, we comparatively evaluated two isolation workflows using freshly resected human PDAC specimens: (i) mechanical explant-based outgrowth and (ii) combined mechanical-enzymatic dissociation with trypsin pre-digestion. In parallel, we evaluated graded antibiotic and antimycotic regimens to optimize contamination control during the initial culture period. The addition of amphotericin B to the culture medium effectively reduced contamination risk without compromising fibroblast outgrowth when withdrawn in a stepwise manner. Furthermore, gentle enzymatic pre-digestion enhanced overall isolation efficiency without affecting the timing of initial fibroblast outgrowth. Phenotypic characterization confirmed fibroblast identity and demonstrated comparable expression of activation-associated markers, although inter-sample variability was observed in signaling receptor expression, including PDGFR- α and PDGFR- β . Collectively, these findings establish a practical and reproducible protocol for isolating primary PDAC-derived CAFs suitable for downstream experimental studies of the tumor microenvironment.
Sabová et al. (Tue,) studied this question.