Long-term cryopreservation of hematopoietic stem and progenitor cells: functional viability beyond two decades.

Due to their ability to differentiate into the full diversity of blood cells, CD34+ hematopoietic stem and progenitor cells (HSPC) can be used to treat a variety of diseases. In autologous transplantation CD34+HSPC are harvested when patients are in remission, cryopreserved and later reinfused after therapy. In many cases the harvested cells are cryopreserved for several years. It is unclear how extended, long-term cryostorage affects the quality of CD34+HSPC. This study assessed quality markers in a unique library of 30 CD34+HSPC grafts, cryopreserved for up to 34 years. The samples were divided into 3 groups (<10y, 10-19y and ≥20y) based on their time in cryostorage. Viability indicators, phenotypic as well as functional markers were evaluated. Our results concluded that CD34+HSPC cryostored grafts were resilient to time. No quality marker, except production of selected cytokines, differed between the first and second decade of preservation. After more than two decades of preservation the viability of total leukocytes (CD45+7-AAD-) (P = 0.041), HSPC (CD34+7-AAD-) (P = 0.015), the functionality measured by CFU (P = 0.005) and Th1 and Th2 cytokine production of the grafts were significantly decreased. Despite this, grafts preserved more than twenty years, retained some viability and some ability to form colonies. In addition, most of the live cells retained enzymatic function and capacity to produce cytokines. In conclusion, although more cells die with time, the cells surviving cryopreservation retain some functional capacity after more than two decades of storage. Based on this study, it is hard to identify a time-limit for cryostorage.