Department of Haematology, PLA Navy General Hospital, Beijing 100048, China.
Cryobiology. 2012 Jun;64(3):167-75. doi: 10.1016/j.cryobiol.2012.01.004. Epub 2012 Jan 18.
To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs).
Samples of human BMCs that were cryopreserved for 23-25 years (n=20) were thawed to obtain an initial culture and a primary culture (P(0)) that was propagated through five passages (P(1)-P(5)) to obtain MSCs. Freshly collected human bone marrow samples (n=20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P(3) cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing.
In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p<0.05) while the relative rate of recovery of MSCs was only 48.5±8.6% in P(0). At the end of P(3), fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p>0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P(3) from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells.
Using the methods described here, long-term (23-25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.
评估长期冷冻保存的人骨髓细胞（BMCs）作为功能性间充质干细胞（MSCs）的来源。
解冻冷冻保存 23-25 年的人 BMC 样本（n=20）以获得初始培养物和通过五个传代（P(1)-P(5)）进行增殖以获得 MSCs 的原代培养物（P(0)）。新鲜采集的人骨髓样本（n=20）用作对照，以比较 MSCs 回收率和生长特性的效率。对 P(3)培养物进行检测，以确定其分化为成骨细胞、脂肪细胞和神经元细胞的能力。采用适当的染色、免疫组织化学和生化方法在不同培养阶段确定细胞类型。
在初始培养中，冷冻保存细胞的细胞黏附率明显低于对照组（19.7%比 38.2%，p<0.05），而 P(0)中 MSC 的相对回收率仅为 48.5±8.6%。在 P(3)期末，两组中均有大约 95%的细胞呈成纤维细胞样（p>0.05）。这些细胞对 MSC 表面分子（CD90、CD105、CD166、CD44、CD29、CD71、CD73）呈阳性，对造血和内皮细胞标志物（CD45、CD34、HLA-DR）呈阴性。两组细胞的生长和细胞周期模式相似。两组 P(3)的 MSCs 均具有相似的体外向成骨细胞、脂肪细胞和神经元细胞分化的能力。
使用此处描述的方法，可以成功地培养长期（23-25 年）冷冻保存的人 BMC 以获得具有良好分化能力的 MSCs。
