Analysis of cord blood CD34+ cells purified after cryopreservation.

Many practical issues regarding processing blood samples for cord blood banking remain. After cryopreservation, a reduction in clonogenicity has been reported, although it is unknown whether this is associated with lower potential for long-term engraftment. CD34+ cell purification of cryopreserved cord blood (CB) may be important for the clinical application of in vitro expansion. We compared purity, yield, clonogenicity, and growth in long-term stromal-based culture of fresh and cryopreserved CD34+ purified cells (n = 12) using the miniMACS separation system. Mean purity of CD34+ cells was 93% when processed before and 73% when processed after cryopreservation. Fresh CD34+ cells had higher clonogenic potential than cryopreserved cells (45 vs 20%, p < 0.05) in CFU-Mix assays, indicating that progenitor cell loss during cryopreservation is due in part to reduced cloning efficiency of viable CD34+ cells. In long-term culture (LTC) on irradiated normal human bone marrow stroma (n = 7), CFU-GM production in the two groups was the same over 12 weeks, suggesting identical long-term culture-initiating cell (LTC-IC) numbers. We conclude that apparent clonogenic cell loss during cryopreservation is associated with relative sparing of the more primitive LTC-ICs. CFU-Mix assays may therefore underestimate the transplant potential of cryopreserved CB. Purification of CD34+ cells following cryopreservation gives sufficient purity for detailed evaluation of CD34+ cells and for stem cell expansion.