Influence of G-CSF Mobilization and Liquid Storage Time on Post-Thaw Recovery of Lymphocyte Subpopulations and CD34+ Cells in Cryopreserved Apheresis Products

Background: Peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) for allogeneic transplantation of stem cells or donor lymphocyte infusion are frequently cryopreserved to allow cellular therapy at later time-points. Differences in the post-thaw recovery of CD34⁺ and CD3⁺ T cells in cryopreserved products have been hypothesized to depend on liquid storage time or, in the case of CD3⁺ T cells, on the mobilization by G-CSF.
Methods: The recovery of CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺, CD16⁺CD56⁺, and CD34⁺ cells from 58 allogeneic apheresis products (among which 38 derived from G-CSF-mobilized donors) was analyzed by flow cytometry in order to evaluate the impact of the freezing/thawing process in the recovery of lymphocyte subpopulations as well as hematopoietic stem cells. In addition, cell viability was determined by measuring viable CD45⁺ (vCD45⁺) cells. All measurements were performed with aliquots that had been cryopreserved in parallel with the products using a controlled-rate freezer and 8% dimethyl sulfoxide and were stored in liquid nitrogen.
Results: The post-thaw recovery was 78.6 ± 14.7% (mean±SD) for CD3⁺CD4⁺ cells, 79.0 ± 12.9% for CD3⁺CD8⁺ cells, 95.6 ± 15.8% for CD19⁺ cells, 84.6 ± 22.4% for CD16⁺CD56⁺ cells, and 93.9 ± 24.6% for CD34⁺ cells. In G-CSF mobilized products, higher recovery rates were observed than in non-mobilized products, reaching statistical significance for CD3⁺CD4⁺ T cells (82.8 ± 14.5 vs 72.1 ± 12.8%, p=0.008), CD19⁺ B cells (99.3 ± 15.9 vs 89.7 ± 14.1%, p=0.027), and CD16⁺CD56⁺ NK cells (90.1% ± 19.8% vs 75.9 ± 22.4%, p=0.025). Within the lymphocyte subpopulations the post-thaw recovery was significantly lower for CD3⁺CD4⁺ vs CD19⁺ (p=1.0x10^-9 in G-CSF mobilized products, p=2.9x10^-5 in non-mobilized products), and CD3⁺CD8⁺ vs CD19⁺ (p=1.8x10^-8 in G-CSF mobilized products, p=3.2x10^-4 in non-mobilized products). With a ratio of vCD45⁺/CD45⁺ of 62.6 ± 18.2% in G-SCF mobilized products and 64.4 ± 18.3% in non-mobilized products no difference in the viability was observed. Spearman’s analysis revealed only a weak negative correlation between liquid storage time (30 ± 14 h for G-CSF mobilized products, 22 ± 7 h for non-mobilized products) and viability (r_s=-0.33, p=0.015), and between liquid storage time and CD34⁺ recovery (r_s=-0.42, p=0.009). The post-thaw recovery of all other cell types did not decrease with longer liquid storage time.
Conclusion: G-CSF mobilization and longer liquid storage time do not impair post-thaw recovery of lymphocyte subpopulations when compared to products from non-mobilized donors. However there is a slight decrease of viability with longer liquid storage time. Our results further suggest that T lymphocytes exhibit a higher sensitivity toward freezing and thawing than B lymphocytes, which may have clinical implications for cellular therapies using frozen products.