Bioengineering Laboratory, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA, USA.
La Cell LLC, Tulane University School of Medicine, New Orleans, LA, USA.
Sci Rep. 2018 May 25;8(1):8162. doi: 10.1038/s41598-018-26546-7.
Over the last decade and half, the optimization of cryopreservation for adipose tissue derived stromal/stem cells (ASCs) especially in determining the optimal combination of cryoprotectant type, cooling rate, and thawing rate have been extensively studied. In this study, we examined the functionality of ASCs that have been frozen-stored for more than 10 years denoted as long-term freezing, frozen within the last 3 to 7 years denoted as short-term freezing and compared their response with fresh ASCs. The mean post-thaw viability for long-term frozen group was 78% whereas for short-term frozen group 79% with no significant differences between the two groups. The flow cytometry evaluation of stromal surface markers, CD29, CD90, CD105, CD44, and CD73 indicated the expression (above 95%) in passages P1-P4 in all of the frozen-thawed ASC groups and fresh ASCs whereas the hematopoietic markers CD31, CD34, CD45, and CD146 were expressed extremely low (below 2%) within both the frozen-thawed and fresh cell groups. Quantitative real time polymerase chain reaction (qPCR) analysis revealed some differences between the osteogenic gene expression of long-term frozen group in comparison to fresh ASCs. Intriguingly, one group of cells from the short-term frozen group exhibited remarkably higher expression of osteogenic genes in comparison to fresh ASCs. The adipogenic differentiation potential remained virtually unchanged between all of the frozen-thawed groups and the fresh ASCs. Long-term cryopreservation of ASCs, in general, has a somewhat negative impact on the osteogenic potential of ASCs, especially as it relates to the decrease in osteopontin gene expression but not significantly so with respect to RUNX2 and osteonectin gene expressions. However, the adipogenic potential, post thaw viability, and immunophenotype characteristics remain relatively intact between all the groups.
在过去的十年半时间里，人们对脂肪组织来源的基质/干细胞（ASCs）的冷冻保存进行了广泛的优化研究，特别是在确定最佳的冷冻保护剂类型、冷却率和复温率组合方面。在这项研究中，我们检查了冷冻保存时间超过 10 年的 ASC（称为长期冷冻）、冷冻保存时间在 3 至 7 年内的 ASC（称为短期冷冻）的功能，并将其与新鲜 ASC 的功能进行了比较。长期冷冻组的解冻后存活率平均值为 78%，短期冷冻组为 79%，两组间无显著差异。对基质表面标志物 CD29、CD90、CD105、CD44 和 CD73 的流式细胞术评估表明，在所有冷冻-解冻 ASC 组和新鲜 ASC 中，P1-P4 代的表达均超过 95%，而造血标志物 CD31、CD34、CD45 和 CD146 的表达在冷冻-解冻和新鲜细胞组中均极低（低于 2%）。实时定量聚合酶链反应（qPCR）分析显示，与新鲜 ASC 相比，长期冷冻组的成骨基因表达存在一些差异。有趣的是，短期冷冻组的一组细胞的成骨基因表达明显高于新鲜 ASC。所有冷冻-解冻组与新鲜 ASC 之间的脂肪分化潜力几乎没有变化。一般来说，长期冷冻保存 ASC 对 ASC 的成骨潜能有一定的负面影响，特别是与骨桥蛋白基因表达的降低有关，但与 RUNX2 和骨粘连蛋白基因表达的降低关系不大。然而，在所有组之间，脂肪分化潜力、解冻后存活率和免疫表型特征仍然相对完整。
