Cryopreservation of hematopoietic stem/progenitor cell (HPC) grafts and other cell based products to be infused in the future is a common practice. However, cryopreservation and conditions of storage can influence post thaw recovery of cellular products. HPC graft is composed of heterogeneous blood cells. Various assays detect viability based on different principles and their sensitivity differs. Less than 1 in 100,000 CD34+ cells possess in vivo hematopoietic reconstitution potential, therefore determination of viability and recovery need to be interpreted accordingly. We analyzed total 38 graft aliquots stored in liquid nitrogen (auto 10 yrs = 7, Auto 1 yr = 12, allo 10 yrs = 5, Allo 5 yrs = 5, allo 1 yr = 9). The viable CD34+ cell frequency in allo HPC grafts was higher than auto grafts cryopreserved 10 years ago (Auto 10 yrs = 0.39 ± 0.24%, Allo 10 yrs = 0.96 ±0.31%, Allo 5 yrs = 0.82 ± 0.3%, p = 0.018). When we compared both single and dual ISHAGE platform methods it displayed comparable post thaw viable CD34+ cell numbers (Figure 1A). The recovery of viable total nucleated cells (TNC) based on trypan blue dye exclusion was 46.83± 4.98% auto 10 years, 43.11 ± 7.76% allo 10 years, 48.74 ± 4.94% allo 5 years, 87.56 ± 6.93% allo 1 year. The viable CD34+ cell recovery between the groups were comparable (Figure 1B). Auto HPC grafts stored for more than 10 years displayed 64.05±16% while auto grafts stored less than a year yielded 85.4±15.15% viable CD34 recovery. The effects of storage duration on grafts is presented below by monitoring hematopoietic engraftment after transplantation. Interestingly, auto grafts in comparison to allo grafts stored for 5 to10 years displayed higher red fraction (JC-1); indicative of higher mitochondrial membrane potential (MMP) and viability (Auto 10 yrs = 72.24 ± 12.45%, Allo 10 yrs = 40.6 ± 13.92%, Allo 5 ysr = 47.2 ± 15.36%, p = 0.011). The higher fraction of cells with intact MMP (red fraction) within CD34+ cell compartment of post thaw suggesting likely higher resilience of auto grafts. Samples stored up to10 years displayed positive colony forming unit (CFU) growth but had poor CFU recovery. CFU recovery for HPC grafts stored for less than 1 year displayed 82.93 ± 25.68% for auto grafts while allo graft displayed 72.46 ± 34.76%. HPC grafts stored for 5 to 10 years which were utilized here to determine graft stability were transplanted within two months of storage and displayed timely neutrophil and platelet engraftment. When single ISHAGE platform method was used to enumerate viable CD34+ cells and HPC grafts are thawed within one year, viable CD34 recovery for auto HPC graft was 85.4 ± 15.15% and for allo graft recovery was 86.85 ±18.0%. Four patients which were transplanted (CD34+ cells infused mean ± SD, 4.35 ± 1.43 x 10e6/KG body weight) with autologous grafts stored for 5 to 8 years in liquid nitrogen (vapor phase) displayed timely neutrophil (9.5 ± 1 day) and platelet (16.5 ± 5 day) engraftment. Taken together, viability assays detect various aspects of cell integrity. The post thaw viable CD34 recovery corresponds well with in vivo hematopoietic reconstitution but in some instances functional potency assays particularly for samples stored longer than 5 years may be crucial. The relatively higher resilience of auto HPC grafts stored for longer than10 years is of note which will need to be further validated.
Patel:Celgene: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Consultancy, Speakers Bureau.
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