Information about donors and tissues is presented in
Supplementary Table 1.
Figure 1 illustrates the acquisition, generation, and culture of prostate TSCs. Radical prostatectomy specimens were obtained immediately post-surgery and 8-mm diameter cores of putative benign and PCa regions were taken from the peripheral zones according to needle biopsy maps and gross analysis (
Figure 1A, B). The cores were aseptically precision-cut to 300-µm thickness in the Krumdieck Tissue Slicer (Alabama Research and Development, Mundford, AL, USA),
8 , 29 from which they emerged in a sequential manner (
Figure 1C, D). Alternating slices were formalin-fixed for paraffin-embedding or frozen and sectioned for histological verification by hematoxylin and eosin (H&E) staining (
Supplementary Figure 1). Adjacent slices were transferred with sterile forceps on to titanium mesh inserts in 6-well plates with 2.5 mL of culture medium and one to five tissue slices per well (
Figure 1E). The plates were incubated at 37°C with 95% air/5% CO
2 on a rotating platform set at a 30° angle (Alabama Research and Development,
Figure 1F). Intermittent submersion in the medium caused by the angled rotation facilitates nutrient and gas diffusion throughout the 300-µm slices (
Figure 1G), which is critical for maintaining cell viability over time. M199, Keratinocyte Serum-Free Medium (KSFM), and antibiotic/antimycotic were from Gibco Life Technologies (Grand Island, NY, USA) and were prepared as in Blauer
et al.
19 (link) Complete PFMR-4A was prepared as previously reported.
30 Dihydrotestosterone (DHT, Steraloids Inc., Newport, RI, USA) and R1881 (Perkin Elmer, Waltham, MA, USA) stocks were prepared in ethanol (Sigma-Aldrich, St. Louis, MO, USA). Piperlongumine (Indofine Chemical Company Inc., Hillsborough, NJ, USA) was prepared in dimethyl sulfoxide (DMSO, Fisher Scientific Inc., Hampton, NH, USA).
Maund S.L., Nolley R, & Peehl D.M. (2013). Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate. Laboratory investigation; a journal of technical methods and pathology, 94(2), 208-221.