离心管贴壁细胞对人脐血单个核细胞体外培养成功率的影响

背景：在常规的脐血间充质干细胞密度梯度离心分离过程中，离心管壁有大量单个核贴壁细胞，这些早期的贴壁细胞是否与脐血间充质干细胞的体外培养成功率低存在一定的联系呢？目的：比较4种方法分离人脐血单个核细胞的体外培养成功率，观察离心管贴壁细胞对人脐血单个核细胞体外培养成功率的影响。方法：取足月健康顺产新生儿脐血36份，随机分为4组，每组9份，于采集后6 h内分别采用甲基纤维素沉降法(A组)、密度梯度离心分离法(B组)、甲基纤维素联合密度梯度离心分离法(C组)、甲基纤维素联合改良密度梯度离心分离法(D组)分离出单个核细胞，锥虫蓝染色检测细胞活力，用细胞计数板进行细胞计数，调整细胞浓度为1×10⁹ L-1~2×10⁹ L^-1，接种于含体积分数为10%胎牛血清的低糖DMEM培养基中培养和传代，相差显微镜下观察脐血单个核细胞的形态。收集第3代脐血单个核细胞，采用细胞计数板进行计数，计算扩增倍数，采用流式细胞仪鉴定细胞免疫表型。结果与结论：36份脐血中，成功分离33份脐血单个核细胞，B组有3份分离失败。33份中共22份原代培养中出现大量贴壁细胞，其中A组8份，B组2份，C组5份，D组7份。9份(27%)培养出能融合且可稳定传代的成纤维样细胞，其中A组3份，B组0份，C组1份，D组5份。4组脐血单个核细胞在原代培养5~7 d后均可见数量不等的贴壁细胞，呈梭形成纤维样细胞或(和)圆形巨核样细胞。A组与D组于原代培养三四周可见成纤维样细胞集落形成，细胞形态与骨髓间充质干细胞相似，呈较均一的长梭形，可稳定培养至60~90 d形态无明显变化。流式细胞仪免疫检测，第3代脐血单个核细胞不表达或弱表达CD34、CD45和CD106等造血干细胞和内皮细胞标志，但显著表达CD29、CD105等间充质干细胞表面标志。结果显示离心管贴壁细胞可显著提高脐血单个核细胞的体外培养成功率，而甲基纤维素联合改良的密度梯度离心分离法，可充分回收离心管贴壁细胞，利于脐血单个核细胞的体外扩增和稳定传代。

Effect of the cells attached to centrifugation tubes on the successful culturing rate of human umbilical cord blood mononuclear cells in vitro

BACKGROUND: A large number of mononuclear cells adhere to the centrifugation tube in the process of densitygradient centrifugation and isolation for the umbilical cord blood mononuclear cells (UCB-MNCs), the correlation between these adherent cells and the success rate of culturing UCB-MNCs in vitro remains unclear.OBJECTIVE:To compare the achievement ratio of cultivating UCB-MNCs in vitro by four different methods and to elucidate the influence of the cells adhering to the centrifugation tube on the cultivating rate of UCB-MNCs in vitro. METHODS:A total of 36 samples were obtained from umbilical cord blood of term deliveries and were divided randomly into four groups, with 9 samples in each. A group: methylcellulose isolation method; B group: density gradient centrifugation isolation method; C group: methylcellulose isolation method plus density gradient centrifugation; D group: methylcellulose isolation method plus modified density gradient centrifugation. Mononuclear cells were separated by the above four methods within 6 hours after collection, seeded in 25 cm2 culture flask at a concentration of 1×10⁹/L - 2×10⁹/L and suspended in low glucose-DMEM cell culture medium supplemented with 0.1 volume fraction of fetal bovine serum. The morphology was observed under contrast phase microscope. UCB-MNCs at passage 3 were collected and counted with cell counting plate. The amplification times were calculated. Flow cytometry was used to examine the surface antigen phenotype of the third passage cells. RESULTS AND CONCLUSION:There were 33 samples successfully isolated out of 36 UCB-MNCs samples, while three samples failed in B group. A large number of adherent cells were seen in 22 of 33 UCB samples (8 samples in Group A; 2 samples in Group B; 5 samples in Group C; 7 samples in Group D). Fibroblastoid cells from 9 samples (3 samples in Group A; 0 sample in Group B; 1 sample in Group C; 5 samples in Group D) were passaged steadily with the achievement ratio of 27.27%. Fibroblast-like cell and round megakaryocyte were the two basic types of adherent cells at 5-7 days after primary culture. At 3-4 weeks after serial subcultivation, UCB-MSCs could be found in A group and D group, they were morphologically similar to MSCs, showing homogeneous fusiform shape. These cells were cultured steadily for 60-90 days without obvious differences in morphology. Flow cytometry showed that, MSCs-related antigens such as CD29 and CD105 were expressed strongly on the surface of UCB-MSCs, but few hematopoietic lineage markers such as CD34 and CD45 nor endothelial markers such as CD106 could be found. Cells adhering to the centrifugation tubes may elevate significantly the achievement ratio of UCB-MSCs in vitro, methylcellulose isolation method combined with modified density gradient centrifugation could fully recover the adherent cells, thus benefiting for amplifying and passaging UCB-MNCs steadily in vitro.