Background & Aims Patient-derived gastrointestinal organoids are powerful tools for studying human physiology and disease. However, generating organoids requires prompt isolation and culture of fresh tissue, which is labor and time intensive, placing restraints on basic and clinical research. For example organoid biobanking efforts, or the ability to obtain specimens from distant locations that lack the expertise to culture organoids, is limited by current approaches. We sought to develop a method by which tissue biopsies from patients could be cryo-preserved, stored, or shipped, and later thawed in order to establish live organoid cultures. Methods A method for freezing and recovery, along with straightforward isolation methods using readily available materials were developed. Epithelial isolation and culture conditions were optimized to promote cell survival and the establishment of long-term culture post-thawing. Results Patient biopsies from stomach, duodenum, ileum, colon and from adenomateous colonic polyps were frozen, subsequently thawed and used to successfully establish patient-specific epithelium-only organoid cultures with 100% efficiency (n=31 independent patient biopsies from different regions of the GI tract). Frozen tissue could be shipped internationally and across the United States and used to establish successful organoid cultures. Organoid cultures could be expanded, passaged and frozen down for long-term storage. RNA-sequencing demonstrates that organoids derived from fresh tissue or from frozen biopsies are >99% transcriptionally identical. Conclusions Cryo-preservation of human tissue biopsies followed by the establishment of long-term, expandable cultures has negligible effect on transcription when compared to freshly prepared organoids, and will be of immense benefit to research and for clinical applications. This technique will allow for the establishment of biobanks of human tissues that can be revived and cultured in the future as well as transported across the globe for research, therapeutic or diagnostic use in remote labs and/or clinics.