Park Seah, Lee Dong Ryul, Nam Ji Sun, Ahn Chul Woo, Kim Haekwon
Department of Biotechnology, Seoul Women's University, 621 Hwarangro, Nowon-Gu, Seoul, Republic of Korea.
Department of Biomedical Science, CHA University, 120 Haeryong-ro, Pocheon-shi, Gyeongghi-do, Republic of Korea.
Cryobiology. 2018 Apr;81:65-73. doi: 10.1016/j.cryobiol.2018.02.008. Epub 2018 Feb 13.
The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (MeSO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches.
由于存在疾病传播风险以及移植宿主中的异种免疫反应，使用胎牛血清（FBS）作为冷冻保存补充剂不适用于间充质干细胞（MSC）的储存。在此，我们研究了人血清白蛋白（HSA）、人血清（HS）或敲除血清替代物（KSR）是否可以替代FBS用于MSC的冷冻保存。此外，我们还检测了多轮冷冻保存后MSC的特性。用三种FBS替代物（9% HSA、90% HS或90% KSR）与10%二甲基亚砜（MeSO）联合冷冻保存的人脂肪来源干细胞（ASC）维持了干细胞特性，包括生长、免疫表型、基因表达模式以及分化为脂肪生成、成骨和成软骨谱系的潜力，这与用FBS冷冻保存的ASC相似。此外，ASC的免疫表型、基因表达和分化能力在多达四个冻融循环后没有改变。然而，如较长的群体倍增时间和降低的集落形成单位-成纤维细胞频率所示，三个或四个冻融循环显著降低了ASC的增殖能力。总之，我们的结果表明，HSA、HS或KSR可以替代FBS用于ASC的冷冻保存，而不会改变其干性，并且在临床应用中冻融循环不应超过两次。
