Journal: Frontiers in Cell and Developmental Biology
Article Title: Optimised dissociation and multimodal profiling of prostate cancer stroma reveal fibromuscular cell heterogeneity with clinical correlates
doi: 10.3389/fcell.2025.1653780
Figure Lengend Snippet: Viable cell yields differ widely between distinct tissue dissociation protocols. (A) Schematic comparison of the tissue dissociation protocols employed demonstrating: common tissue pre-processing steps; enzymatic dissociation with enzyme cocktail composition and incubation times; post-enzymatic dissociation downstream processing steps; and quality assessment via cell viability analysis per trypan blue staining and cell seeding. Readouts are exemplified in (B–D) . (B–E) Analysis of benign tissue cores dissociated using the indicated protocol. (B) Viable cell yield/mg tissue for each protocol. Bars represent the median ± interquartile range from 5, 6 or 29 biological replicates for the Miltenyi and BD 30 min, BD 60 min or optimised protocols, respectively. Statistical significance was determined using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 correction for multiple comparisons. (C) Brightfield imaging for trypan blue viability assessment of freshly dissociated cells isolated using the indicated protocol. Scale bars represent 200 μm at ×10 magnification. (D) Brightfield images of the 10% seeded cells after 4 weeks. Scale bars represent 500 μm at ×4 magnification. Frequency of successful mesenchymal cell growth for each of the four replicates is indicated. (E) Immunofluorescent staining of successfully cultured cells for the epithelial marker pan-cytokeratin (panCK) and mesenchymal markers vimentin and CD90, whereby font colour denotes pseudocolouring in the displayed images. Nuclei were counterstained using Hoechst 33342 (blue). 22Rv1 prostate cancer cells and explant cultures of primary human prostate fibroblasts served as negative and positive controls for panCK or vimentin and CD90, respectively. Scale bars represent 100 μm at ×20 magnification.
Article Snippet: The 22Rv1 prostate cancer cell line was obtained from the American Type Culture Collection (ATCC Nr. CRL-2505; ATCC; Rockville, MD), STR validated and maintained according to the distributor’s instructions.
Techniques: Comparison, Incubation, Staining, Imaging, Isolation, Cell Culture, Marker