Selective cell survival and changes of marker properties during cryopreservation for up to 16 years.

Cell features that may favor resistance to cryoinjury were investigated. Subpopulations of all tissue bank deposits tested (5 human cell lines and 19 mouse tumors) survived up to 16 years of storage at —78°. The replicating capacity of trypan blue-excluding cells after prolonged cryopreservation was estimated by comparing the number of unstained cells needed to produce < 100% lethal takes with control values determined prior to freezing. Frozen-thawed bank deposits were reestablished in serial transplantation or in vitro, without obvious change in virulence or growth rate. Metho trexate tolerance in a drug-selected tissue culture of mouse Sarcoma 180 persisted at its prefreeze resistance level. Host strain specificity, an index of H-antigen stability, was lessened after freezing in only one of five different ascites lymphomas examined. The 6C3HED lymphoma, formerly restricted to growth in the C3H mouse of origin, continued to regress in three foreign strains but produced a few long-delayed lethal takes in three other strains with rela tively low allograft resistance. In one case, this was accompanied by polyploidization. While modal chromo some numbers generally remained quite stable, the frequen cies of characteristic marker chromosomes were altered in two of three murine carcinoma clones and in two lym phomas. These immunological and cytological changes observed in some old bank deposits were not yet evident during the initial 4 years ofstorage at —78°. Two polyploid sublines derived by endoreduplication and immunoselection prior to freezing from the near-diploid ELD carcinoma and 6C3HED lymphoma, as well as four heteroploid clones isolated by transplantation of single cells from the near-tet raploid Ehrlich-KL and Krebs 2 carcinomas, all showed significantly better long-term cryotolerance than the corre sponding source tumors. Most neoplasms may be safely stored at —78° for 4 to 5 years without differential cell mortality and consequent shifts in nuclear and physiological properties.