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.

Keywords: ALDH; cryopreservation; haematopoietic stem cell; mitochondrial activity; pH; potency; storage; viability.

Figure 1.

Determination of viability, necrosis and apoptosis. Detection of viable and apoptotic cells with 7-Aminoactinomycin/Annexin V. (A) A total leukocyte viability in groups; (B) mononuclear cells (MNC) viability, (C) proportion of dead CD34+ cells, (D) percentage of apoptotic cells within the CD34+ cell population. (median; box: 25%–75%; quantiles; * significancy P ≤ 0.05).

Figure 2.

Evaluation of mitochondrial potential. Detection of mitochondrial potential using the JC-1 assay. Percentage of green cells indicates the health of mitochondria in groups. There is no significant difference between native and both groups of cryopreserved cells. (median; box: 25%–75%; quantiles; * significancy P ≤ 0.05).

Figure 3.

Testing of metabolic activity. ALDH expression by CD34+ cells showing metabolic activity of cells. No difference between groups was observed. (median; box: 25%–75%; quantiles; * significancy P ≤ 0.05).

Figure 4.

Analysis of clonogenic potential. The level of ATP/cell measured after activation by HALO^® Culture Master Mix. All groups showed potential to differentiate/proliferate; however, long-term storage slightly decreased the clonogenic potential when compared to native cells. (median; box: 25%–75%; quantiles; * significancy P ≤ 0.05).

Figure 5.

Intracellular pH detection There was a trend for lower intracellular pH (ipH) in the LTC group in comparison to the N subgroup and the STC subgroup but without statistical significancy. (median; box: 25%–75%; quantiles; * significancy P ≤ 0.05).