Differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation －Comparison among cells stored for 1, 5, 10, 15, and 20 years.
Differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation －Comparison among cells stored for 1, 5, 10, 15, and 20 years.
复制标题
DOI：
10.1016/j.reth.2020.01.006
复制
发表时间：
2021-12
影响因子：
4.3
通讯作者：
Takeda A
中科院分区：
文献类型：
--
作者：
Sugimoto Y;Yamazaki Y;Moriyama K;Sugimoto T;Kumazawa K;Baba K;Sone Y;Takeda A
我们研究了长期冷冻保存的人骨组织来源的间充质间质细胞（hBT-MSCs）的骨分化和增殖能力。我们确定了由于冷冻引起的任何形态学和特征变化的存在，以确定未来临床应用需要解决的问题。本研究从我科唇腭裂二次牙槽骨移植患者的多余髂骨松质骨标本中收集15份hBT-MSCs，冷冻保存时间从1年到20年不等，各3份，解冻后再培养。在共聚焦显微镜下观察calcein/ ethd染色细胞的生存能力，并采用水溶四氮唑盐（Water Soluble Tetrazolium salts， WST）法进行一周的细胞增殖实验。共聚焦显微镜还用于鉴定任何过度积累的衰老相关生长因子SA-βgal。分化能力分为以下三组：骨分化、脂肪细胞分化和非分化诱导。在连续培养细胞四周后，我们使用茜素红染色、钙定量和油红染色检测骨/脂肪细胞的分化能力。活力测试结果表明，随着低温保存年限的增加，活细胞比例降低。细胞增殖实验表明，低温保存时间较短的细胞呈指数增长。在老化测试中，超低温保存≥5年的细胞表现出类似的阳性反应，与超低温保存年数无关。在细胞增殖试验中，不同年份的冷冻保存之间无统计学差异。我们将骨分化和脂肪细胞分化能力与非诱导组进行比较，证实诱导组具有统计学优势。而诱导组在不同年龄间无显著差异。冷冻保存20年的样本在骨和脂肪细胞分化方面仍然有能力。然而，它们的分化方向倾向于向骨细胞或脂肪细胞分化。我们的研究结果表明，冷冻不会加速衰老，长期冷冻保存的样品在未来的临床应用中是有用的。
We investigated bone differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation. We determined the presence of any morphological and characteristic changes due to freezing to identify issues that need to be solved for future clinical applications. A total of 15 samples of hBT-MSCs that had been cryopreserved for different lengths of time, ranging from one year to 20 years (n = 3 each), were thawed and recultivated after being collected from excess iliac cancellous bone specimens of patients who underwent secondary alveolar bone grafting for cleft lip and palate in our department. We determined viability by observing calcein/EthD-stained cells under a confocal microscope, and the cell proliferation experiment was performed for one week using the Water Soluble Tetrazolium salts (WST) assay method. A confocal microscope was also used to identify any excessively accumulated senescence-associated growth factor SA-βgal. Differentiation potency was assessed in the following three groups: bone differentiation, adipocyte differentiation, and nondifferentiation induction. We examined bone/adipocyte differentiation potencies using Alizarin Red staining, Ca quantitation, and Oil Red staining after continuously culturing cells for four weeks. Viability test results indicated that the proportion of viable cells decreased as the number of years of cryopreservation increased. The cell proliferation experiment showed that cells cryopreserved for a shorter duration multiplied exponentially. In the aging test, cells cryopreserved for ≥5 years showed similar positive reactions independent of the number of years of cryopreservation. In the cell proliferation test, there was no statistically significant difference between the years of cryopreserving. We compared bone differentiation and adipocyte differentiation ability with the non-induction group, and the induction group was confirmed to have a statistical advantage. However, there was no significant difference in the induction group pertaining to different ages. Samples cryopreserved for 20 years remained competent in bone and adipocyte differentiation. However, their differentiation direction tended to skew to either bone or adipocyte differentiation. Our results suggest that freezing does not accelerate aging, and samples cryopreserved for a long time are useful in future clinical applications.
登录
查看更多内容
影响因子：
3.7
作者：
HAYFLICK, L;MOORHEAD, PS
通讯作者：
MOORHEAD, PS
DOI：
10.1073/pnas.92.20.9363
发表时间：
1995-09-26
影响因子：
11.1
作者：
DIMRI, GP;LEE, XH;CAMPISI, J
通讯作者：
CAMPISI, J
影响因子：
0.9
作者：
Matsuo, Aoi;Yamazaki, Yasuharu;Uchinuma, Eiju
通讯作者：
Uchinuma, Eiju
影响因子：
3.1
作者：
Behnia, Hossein;Khojasteh, Arash;Atashi, Amir
通讯作者：
Atashi, Amir
影响因子：
2.9
作者：
Gary, RK;Kindell, SM
通讯作者：
Kindell, SM