Prostate Tumor Explant Culture Protocol

The PDE technique utilized in this study employs a gelatin sponge platform (Centenera et al., 2013) (Fig. 1A). This method was selected for two key reasons: firstly, the use of a substrate for explant culture prevents cellular outgrowth that is frequently observed when tissues are cultured without support and completely submerged in media (Pretlow et al., 1995; Varani et al., 1999); secondly, the gelatin sponge used in this study is a commercial medical device developed for hemostasis (Ferrosan, 2014) that is readily available, cost‐effective, and simple to use, making the method feasible for widespread adoption in translational cancer research laboratories.
Figure 1A illustrates the PDE method using prostate tumors as an example. Following surgical tissue removal, the surgeon or clinical pathologist resects a sample of the presumed malignant or nonmalignant region and the specimen is transported to the research laboratory in cold phosphate‐buffered saline on ice, typically within 1–2 h of surgery. Under sterile conditions, tissue was placed onto the lid of a 10‐cm plate along with the saline it was transported in. Using a surgical blade, a 1‐mm‐thick longitudinal section of the tissue sample is cut and placed into neutral‐buffered formalin for paraffin embedding. This tissue is called the Day 0 sample and is used to determine the cancer content of the tissue following staining with hematoxylin and eosin (H&E). The remaining tissue is dissected into 1‐mm³ pieces, called explants, and placed in triplicate or quadruplicate (depending on amount of tissue received) on top of a media‐soaked gelatin sponge (Ethicon; Johnson & Johnson, Somerville, NJ, USA) inside the wells of a 24‐well plate containing 500 μL RPMI 1640 medium containing 10% fetal bovine serum, 1× antimycotic/antibiotic solution, 0.01 mg·mL⁻¹ hydrocortisone, and 0.01 mg·mL⁻¹ insulin. The appropriate vehicle, treatment, or shRNA was added directly to the media inside the appropriate tissue culture well at the indicated concentrations, allowing direct comparison of treatments and controls. Explants were cultured at 37 °C and 5% CO₂ for various time points and then formalin‐fixed and paraffin‐embedded, snap‐frozen, or preserved in RNAlater (Invitrogen, San Diego, CA, USA) depending on the desired downstream analysis. For assessment of BrdU incorporation, BrdU (10 μm) was added to the culture medium 2 h (prostate) or 24 h (breast) prior to harvest.
In our collective experience, all tissues containing epithelial cells can successfully be cultured, and the limiting factor for analysis is instead the presence of sufficient numbers of epithelial (for benign tissue studies) or malignant cells (for cancer tissue studies). For this reason, routine hematoxylin and eosin (H&E) staining of all Day 0 and cultured tissues is an essential part of our protocol to confirm the presence and approximate percentage of benign/malignant cells within the specimens before proceeding with further analyses. Between the three prostate cancer laboratories, our collective experience indicates that approximately 10% of tissues from high‐volume disease and 20–30% of tissues from low‐volume disease do not contain malignant cells, and this is largely due to sampling.