The cryopreservation of cancer tissues to generate frozen libraries is a common practice used worldwide for storing patient samples for later applications. However, frozen samples stored by existing methods cannot be used for initiating living cell cultures, such as patient-derived tumor organoids (PDOs), which offer great potential for personalized treatment. To overcome this challenge, we developed a novel procedure for culturing PDOs using frozen live tumor tissues. We show that tumor specimens stored using this technique maintain their viability and can be successfully used to generate organoids even after long-term freezing, with an impressive success rate of 95.2 %. Importantly, we found that the structural features, tumor marker expression, and drug responses of organoids derived from frozen tissues are similar to those derived from fresh tissues. Moreover, organoids derived from frozen tissues can be routinely passaged and frozen, making them ideal for high-throughput drug screening at any time. Notably, cryopreserved tumor tissues can also be utilized in air-liquid interface (ALI) culture. This method allows for preserving the original tumor microenvironment, making it an invaluable resource for conducting tests on antitumor drug responses, including immune checkpoint inhibitors (ICIs). This innovation has the potential to enable the identification of potentially effective drugs for patients and facilitate the development of novel therapeutic drugs. Thus, we have established protocols for the long-term cryopreservation of cancer tissues to maintain their viability and microenvironment, which are useful for personalized therapy.© 2024 The Authors.