Expansion of multilineage prostate organoids derived from human tissue in ProstaCult™ organoid medium

Organoids have emerged as a powerful model for studying normal and tumour cell biology; however, the application of organoid technology for culturing human prostate epithelial cells has been challenging due to the difficulty in maintaining multilineage organoids with androgen receptor (AR)-expressing cells in culture for extended periods of time. To address this, we developed serum-free ProstaCult™ Organoid Medium (Human) to robustly expand human prostate epithelial cells long-term as organoids. Benign human prostate tissue was dissociated and seeded either directly into Matrigel® dome cultures or pre-cultured up to 4 passages in adherent culture in EpiCult™ Plus medium prior to seeding. The resulting organoids were passaged as small fragments every 10 - 12 days. Cultures were analyzed by flow cytometry and immunocytochemistry for the expression of pan epithelial marker EpCAM, luminal markers CD26, keratin 8 (K8), and androgen receptor (AR) protein, and basal markers CD271 and K14. Our results demonstrate that prostate epithelial cells can generate multilineage organoids consisting of a polarized epithelium with both basal (EpCAM + , K14 + , CD271 high /CD26 low ) and luminal cells (EpCAM + , AR + , K8 + , CD271 low /CD26 high ) in 10 - 12 days. These human prostate organoids can be expanded for at least 7 passages while maintaining appropriate marker expression (n = 5) and can be cryopreserved without losing long-term expansion potential. The organoids were responsive to dihydrotestosterone (DHT) removal from the culture medium, with a 43 ± 7% decrease in the total number of luminal cells generated over 3 passages (mean ± SEM; n = 4; p ≤ 0.01, paired t-test) and a downregulation of AR expression (n = 4). Similarly, enzalutamide treatment resulted in a 24 ± 9% decrease in the total number of luminal cells generated over 3 passages (mean ± SEM; n = 4). These results demonstrate that ProstaCult™ Organoid Medium (Human) enables robust long-term maintenance of multilineage organoids containing AR+ luminal cells in vitro , providing an optimized and standardized model system for prostate cancer research.