Assessment of cell viability, early apoptosis, and hematopoietic potential in umbilical cord blood units after storage

BACKGROUND: Successful hematopoietic stem cell transplantation using stored umbilical cord blood (CB) largely depends on cell dose and quality of CB units. The aim of this study was to assess the degree of early apoptosis, in addition to cell viability and hematopoietic potential, in umbilical CB units after storage. STUDY DESIGN AND METHODS: Sixty CB units that had been cryopreserved for up to 8 years in a single public CB bank were investigated. After the CB units were thawed, cell viability and early apoptosis of total nucleated cells (TNCs), mononuclear cells (MNCs), and CD34+ cells were determined using flow cytometric method based on 7-aminoactinomycin D (7-AAD) and annexin V staining. Next, clonogenic assays to predict graft potency were performed. RESULTS: Postthawing cell viability values determined by 7-AAD were as follows: TNCs, 78.8% +/- 65.8%; MNCs, 88.4% +/- 65.8%; and CD34+ cells, 94.1% +/- 63.2%. Cell viability values using 7-AAD and annexin V dual staining were as follows: TNCs, 71.2% +/- 611.3%; MNCs, 83.1% +/- 67.0%; and CD34+ cells, 88.8% +/- 66.0%. Early apoptotic cells (7-AAD-negative and annexin V-positive cells) in TNCs, MNCs, and CD34+ cells were 6.4% +/- 63.5%, 5.4% +/- 63.1%, and 5.3% +/- 64.1%, respectively. The corrected colony-forming unit-granulocyte-macrophage content per 100 CD34+ cells was 67.5 +/- 48.7. CONCLUSIONS: Postthawing cell viability determined by flow cytometric methods was in the following order: TNCs< MNCs< CD34+ cells. CD34+ cell viability was nearly identical to that of fresh CB 48 hours after collection. Necrosis or apoptosis in cryopreserved CB units did not accelerate during storage.