ANALYSIS OF CORD-BLOOD CD34(+) CELLS PURIFIED AFTER CRYOPRESERVATION

Many practical issues regarding processing blood samples for cord bloo d 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 purificat ion of cryopreserved cord blood (CB) may be important for the clinical application of in vitro expansion. We compared purity, yield, clonoge nicity, and growth in long-term stromal-based culture of fresh and cry opreserved CD34(+) purified cells (n = 12) using the miniMACS(R) separ ation system. Mean purity of CD34(+) cells was 93% when processed befo re 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 durin g cryopreservation is due in part to reduced cloning efficiency of via ble CD34(+) cells. In long-term culture (LTC) on irradiated normal hum an bone marrow stroma (n = 7), CFU-GM production in the two groups was the same over 12 weeks, suggesting identical long-term culture-initia ting cell (LTC-IC) numbers. We conclude that apparent clonogenic cell loss during cryopreservation is associated with relative sparing of th e more primitive LTC-ICs. CFU-Mix assays may therefore underestimate t he transplant potential of cryopreserved CB. Purification of CD34(+) c ells following cryopreservation gives sufficient purity for detailed e valuation of CD34(+) cells and for stem cell expansion.