Cryopreserved cord blood mononuclear cells in DMSO are healthy for at least 6 hours after thawing

Abstract

We investigated the impact of time, storage temperature, and dimethyl sulfoxide (DMSO) on the viability of HSCs, as well as on apoptotic changes in thawed CB. Materials & Methods: Thirteen units of cryopreserved CB were thawed and half of each sample was stored at room temperature (RT) and the other half at 4℃, without removing or diluting DMSO. Flow cytometry was employed to enumerate total nucleated cells (TNCs), total/viable CD34+ cells, and early/late apoptotic cells using anti-CD45, anti-CD34, and annexin V(AnV), 7-amino actinomycin D(AAD) staining, respectively. Results: In CBs stored at 4℃ there were no significant changes in numbers of TNCs, total/viable CD34 + cells, or early/late apoptotic cell up to 48 h. However, the numbers of these cells declined significantly at RT. Total and viable CD34 + cell counts did not change for up to 6 h at RT but viable CD34+ cells decreased significantly after 24 h, and total CD34+ cell after 48 h. Early and late apoptosis tended to increase with time at RT, and numbers of viable CD34+ cells and early apoptotic cells differed significantly between RT and 4℃ after 48 h. Conclusions: There are no significant changes of viability and apoptosis in CBs stored in DMSO at 4℃ until 48 h after thawing, while at RT, there are no significant changes of total/viable CD34+ cell counts or in the proportion of apoptotic cells for at least 6 h after thawing.

Role of flow cytometry in evaluation of the cellular therapy products used in haematopoietic stem cell transplantation

“…The results of stability studies of thawed UCB samples were conflicting. Lee et al 54 in their study showed that thawed UCB samples were stable up to 6 h after thawing, regardless of whether samples were stored at room temperature or at 4°C. On the other hand, Huang and colleagues reported that CD34+ cell viability decreased significantly after only 20 min when thawed UCB samples were stored at RT 55 .…”

“…Sample stability study is an important part of validation of the protocol used for quality assessment, especially when implementing a new type of cell therapy product. Several studies have examined the stability of fresh leukapheresis samples and thawed cryopreserved UCB samples 37,39,54,55 . The results showed that fresh leukapheresis samples were stable up to 24 h stored at 4°C 37,39 .…”

“…Anti-CD34 Haematopoietic stem cells [14,17,18,23,24,33,34,37,39,43,45,47,48,[54][55][56] Anti-CD3 T cells [38,40,45,47,48,50,52] Anti-CD19 Anti-CD20…”

“…Several studies have examined the stability of fresh leukapheresis samples and thawed cryopreserved UCB samples. 37,39,54,55 The results showed that fresh leukapheresis samples were stable up to 24 h stored at 4 C. 37,39 The results of stability studies of thawed UCB samples were conflicting. Lee et al 54 in their study showed that thawed UCB samples were stable up to 6 h after thawing, regardless of whether samples were stored at room temperature or at 4 C. On the other hand, Huang and colleagues reported that CD34+ cell viability decreased significantly after only 20 min when thawed UCB samples were stored at RT.…”

“…37,39,54,55 The results showed that fresh leukapheresis samples were stable up to 24 h stored at 4 C. 37,39 The results of stability studies of thawed UCB samples were conflicting. Lee et al 54 in their study showed that thawed UCB samples were stable up to 6 h after thawing, regardless of whether samples were stored at room temperature or at 4 C. On the other hand, Huang and colleagues reported that CD34+ cell viability decreased significantly after only 20 min when thawed UCB samples were stored at RT. 55 Krasna et al 56 evaluated the stability of the HSC products after immunoselection and reported that CD34+ cells after selection were less stable than CD34+ cells in leukapheresis product during refrigerated storage up to 6 days.…”