One in six men will be diagnosed with prostate cancer (PCa), making it one of the leading health problems affecting men in today’s society. Patients diagnosed during the earlier stages are surviving longer due to improved therapies and the prevalence of prostate-specific antigen (PSA) testing. However, over 80% of advanced PCa patients develop bone metastatic prostate cancer for which there is no cure (Khan and Partin 2003). Cell lines, widely used for the development of new drug treatments and therapies, fail to accurately represent the heterogeneity of prostate cancer. Thus, it is important to establish new patient-derived cell culture models for bone metastatic prostate cancer which could better recapitulate the physiological processes that occur in vivo. Through the incorporation of previously established methods into our own culturing methods, we optimized three-dimensional cell culture conditions which keep our patient-derived xenograft (PDX) and primary patient tumor cells viable in vitro for a month and a half without passaging. Furthermore, preliminary experiments showed that our three-dimensional cultures consist of heterogeneous cell populations that exhibit different responses to the androgen hormone, dihydrotestosterone (DHT), and the anti-androgen drug, Enzalutamide. In future experiments, we hope to further optimize our culturing conditions to improve the robustness and reproducibility of our three-dimensional cell cultures for patient-derived xenograft and primary prostate cancer tumor cells.