Assessment of Cell Viability and Apoptosis in Human Umbilical Cord Blood Following Storage

Abstract

There is increasing use of human umbilical cord blood (UCB) stem cells for unrelated donor transplantation. Successful engraftment depends in large measure on the dose and quality of cells in the UCB unit. In the present study, we attempted to identify a simple and rapid technique for assessing the quality and recovery of UCB cells following laboratory manipulation. Mononuclear cells (MNC) from fresh (<48 h old) and thawed UCB units were stained with 7-amino-actinomycin D (7-AAD), revealing 2-3% dead cells. The frequencies of apoptotic cells in fresh and thawed sample were similar. However, UCB held for 72 h showed higher levels of cell deterioration. Interestingly, staining with 7-AAD was more sensitive to cellular damage than was uptake of Trypan Blue. 7-AAD staining of MNC also correlated with retention of hematopoietic function (progenitor assays) such that 7-AAD staining frequencies <20% predicted maintenance of hematopoietic cells. Importantly, hematopoietic precursors were less susceptible to storage injury than were UCB MNC as a whole. MNC showed higher levels of 7-AAD staining and apoptosis than did CD34(+) cells. This observation was confirmed in studies of caspase-3 activation, where MNC consistently showed higher frequencies of activation than did CD34(+) cells, especially after overnight storage. Furthermore, antiapoptotic proteins Bcl-2 and Bcl-x were expressed more consistently in CD34(+) cells than in total MNC. In contrast, Bax levels increased with MNC apoptosis. In conclusion, the data suggest that 7-AAD staining of UCB MNC provides a rapid and simple technique for assessing the viability, recovery, and hematopoietic functionality of stored UCB.

Apoptotic lymphocytes and CD34+ cells in cryopreserved cord blood detected by the fluorescent vital dye SYTO 16 and correlation with loss of L-selectin (CD62L) expression

“…These findings are consistent with those reported by others for cryopreserved CB cells. 18,19 Increased expression of antiapoptotic proteins (e.g. Bcl-2 and caspase-8 inhibitor FLIP) compared to pro-apoptotic proteins (e.g.…”

“…Bax and activated caspases) by CD34 þ cells may provide an explanation, at least in part, to the differences seen in cryoinjury susceptibility between CD34 þ cells and lymphocytes. [19][20][21] The greater susceptibility of lymphocytes to cryoinjury is relevant in the context of multiple-unit CB transplants where long-term engraftment corresponds more closely to the CB unit with the highest CD3 þ cell dose rather than the CD34 þ cell content of the CB unit. 22 Accurate enumeration of live lymphocytes, in addition to enumeration of live CD34 þ cells, following thawing of CB products may become an important consideration for prediction of transplant outcome in multi-CB unit transplants.…”

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…The freezing process involves complex phenomena of water crystallization and changes in solute concentration both outside and inside the cell that can be detrimental to cell survival. In addition, exposure to low temperatures has been reported to induce a stress response resulting in biomolecular-based cell death for different cell types (Baust et al, 2001;Fu et al, 2001;Paasch et al, 2004;Xiao & Dooley, 2003).…”

Cryopreservation

“…11,48,53,[64][65][66][67][68] Comparison of the stressors associated with cryopreservation and those known to activate apoptosis reveals substantial overlap. An analysis of these stresses and the cryopreservation literature demonstrates the well documented involvement of apoptosis in cryopreservation failure and the benefits of cell stress response modulation to improve outcome.…”