Kim Kyeong Mi, Huh Ji Young, Hong Sang Sook, Kang Myung Seo
Department of Laboratory Medicine, CHA Bundang Medical Center, CHA University.
CHA Medical Center Cord Blood Bank, Gyeonggi-do, Korea.
Transfusion. 2015 Aug;55(8):2017-22. doi: 10.1111/trf.13120. Epub 2015 Apr 10.
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.
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.
Postthawing cell viability values determined by 7-AAD were as follows: TNCs, 78.8% ± 5.8%; MNCs, 88.4% ± 5.8%; and CD34+ cells, 94.1% ± 3.2%. Cell viability values using 7-AAD and annexin V dual staining were as follows: TNCs, 71.2% ± 11.3%; MNCs, 83.1% ± 7.0%; and CD34+ cells, 88.8% ± 6.0%. Early apoptotic cells (7-AAD-negative and annexin V-positive cells) in TNCs, MNCs, and CD34+ cells were 6.4% ± 3.5%, 5.4% ± 3.1%, and 5.3% ± 4.1%, respectively. The corrected colony-forming unit-granulocyte-macrophage content per 100 CD34+ cells was 67.5 ± 48.7.
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.
使用储存的脐带血（CB）成功进行造血干细胞移植很大程度上取决于CB单位的细胞剂量和质量。本研究的目的是评估储存后脐带CB单位中除细胞活力和造血潜能外的早期凋亡程度。
对在单个公共CB库中冷冻保存长达8年的60个CB单位进行了研究。CB单位解冻后，使用基于7-氨基放线菌素D（7-AAD）和膜联蛋白V染色的流式细胞术方法测定总核细胞（TNC）、单核细胞（MNC）和CD34+细胞的细胞活力和早期凋亡情况。接下来，进行克隆形成试验以预测移植物效力。
通过7-AAD测定的解冻后细胞活力值如下：TNC为78.8%±5.8%；MNC为88.4%±5.8%；CD34+细胞为94.1%±3.2%。使用7-AAD和膜联蛋白V双重染色的细胞活力值如下：TNC为71.2%±11.3%；MNC为83.1%±7.0%；CD34+细胞为88.8%±6.0%。TNC、MNC和CD34+细胞中的早期凋亡细胞（7-AAD阴性且膜联蛋白V阳性细胞）分别为6.4%±3.5%、5.4%±3.1%和5.3%±4.1%。每100个CD34+细胞的校正集落形成单位-粒细胞-巨噬细胞含量为67.5±48.7。
通过流式细胞术方法测定的解冻后细胞活力顺序为：TNC＜MNC＜CD34+细胞。CD34+细胞活力与采集后48小时的新鲜CB几乎相同。冷冻保存的CB单位中的坏死或凋亡在储存期间并未加速。
