BACKGROUND: Cellular immunotherapy exhibits promise in treating blood tumors. However, its application for solid tumors is impeded by their heterogeneity and complex microenvironments. The development of individualized multitarget therapy may be the key to overcoming the challenge of tumor heterogeneity. METHODS: To generate tumor-reactive T cells, we modified the conditional reprogramming primary cell culture method by to establish a primary prostate cancer cell culture approach, refer to as eCR (enhanced conditional reprogramming). Then, Tumor tissue-derived primary cells were physically lysed and loaded into dendric cells, which, in turn, were co-cultured with peripheral blood T cells to induced individualized tumor-reactive T cells. RESULTS: Our improved culture method could use a small amount of fresh or frozen tumor specimens (including biopsy specimens), which can be amplified in vitro while maintaining their original characteristics, without contamination by heterologous antigens. Furthermore, a series of in vitro and in vivo experiments revealed these tumor-reactive T cells exhibited specific and effective killing of tumor cells through their ability to recognize neoantigens in cancer. CONCLUSION: In this study, we developed a protocol for the generation of tumor-responsive T cells based on autologous tumor antigens in patients with prostate cancer. This platform is characterized by its multitargeted, individualized, affordability, and minimal adverse effects, holding significant promise in the treatment of prostate cancer as well as other solid tumors.