Long-Term Cryopreservation Does Not Affect Quality of Peripheral Blood Stem Cell Grafts: A Comparative Study of Native, Short-Term and Long-Term Cryopreserved Haematopoietic Stem Cells

Abstract

Cryopreserved haematopoietic progenitor cells are used to restore autologous haematopoiesis after high dose chemotherapy. Although the cells are routinely stored for a long period, concerns remain about the maximum storage time and the possible negative effect of storage on their potency. We evaluated the effect of cryopreservation on the quality of peripheral stem cell grafts stored for a short (3 months) and a long (10 years) period and we compared it to native products.The viability of CD34+ cells remained unaffected during storage, the apoptotic cells were represented up to 10% and did not differ between groups. The clonogenic activity measured by ATP production has decreased with the length of storage (ATP/cell 1.28 nM in native vs. 0.63 in long term stored products, P < 0.05). Only borderline changes without statistical significance were detected when examining mitochondrial and aldehyde dehydrogenase metabolic activity and intracellular pH, showing their good preservation during cell storage. Our experience demonstrates that cryostorage has no major negative effect on stem cell quality and potency, and therefore autologous stem cells can be stored safely for an extended period of at least 10 years. On the other hand, long term storage for 10 years and longer may lead to mild reduction of clonogenic capacity. When a sufficient dose of stem cells is infused, these changes will not have a clinical impact. However, in products stored beyond 10 years, especially when a low number of CD34+ cells is available, the quality of stem cell graft should be verified before infusion using the appropriate potency assays.

Chemical approaches to cryopreservation

“…These low-molecular-weight cryoprotectants were originally discovered over 60 years ago 5 , 6 (not rationally selected for a particular biological/physical property), yet they have been exceptionally effective and widely used in both research and clinical applications. Stocks of red blood cells are cryopreserved using 20–40 wt% glycerol 7 , immortalized cell lines are routinely stored in 10% (v/v) DMSO and cells for clinical transplantation, including haematopoietic stem cells and chimeric antigen receptor (CAR)-T cells, are frequently cryopreserved in 5–10% DMSO 8 , 9 (amongst other additives).…”

Flow Cytometric Characterization of Hematopoietic Stem and Progenitor Cell Subpopulations in Autologous Peripheral Blood Stem Cell Preparations after Cryopreservation

“…Even though the maximum duration of cryopreservation of HSPC products in our survey was 2.1 years, retention samples were cryopreserved significantly longer. However, previous studies have demonstrated that cryostorage has no clinically relevant negative effect on stem cell quality and potency, even after an extended period of more than 10 years [ 12 , 40 ].…”

Pros and Cons of Cryopreserving Allogeneic Stem Cell Products

“…Most cryopreserved allogenic stem cell products are usually infused within months from harvesting, minimizing the possible additional detrimental effect of long storage on CD34+ quality. Moreover, most of the loss of CD34+ cells tend to occur during pre-storage manipulation and subsequent thawing, as storage for up to 10 years does not significantly affect the number, viability, metabolic function, and clonogenic potential of hematopoietic stem cells [ 53 ]. On the other side, for allogenic products, a significant amount of time may elapse between donation and cryopreservation.…”