Advanced Cellular Therapy Laboratory, Department of Cellular Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy.
Cytotherapy. 2013 Aug;15(8):920-9. doi: 10.1016/j.jcyt.2013.01.219. Epub 2013 Apr 24.
A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.
Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.
After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.
The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.
胎牛血清（FBS）是一种常用于扩增人间充质基质细胞（MSCs）的培养基。然而，由于存在人畜共患病和免疫反应的风险，监管机构不鼓励使用 FBS。通过冷冻/解冻破坏血小板获得的人血小板裂解液（PL）已被提议作为 FBS 的可能替代品。该过程耗时且尚未得到很好的标准化。本文提出了一种新的获得 PL 的方法，该方法基于使用超声。
将含血小板的塑料袋浸入超声浴中进行血小板超声处理。为了评估血小板裂解，我们测量了血小板衍生生长因子-AB 的释放。通过扩增骨髓（BM）-MSCs 来测试 PL 的效率，测量群体倍增时间、分化能力和免疫原性。通过核型分析扩增细胞来评估安全性。
在超声处理 30 分钟后，74%的血小板衍生生长因子-AB 被释放。PL 增强了 BM-MSC 的增殖率，与 FBS 相比。在 10%、7.5%和 5%PL 中细胞生长的平均累积群体倍增（cPD）优于在 10%FBS 中获得的 cPD。与通过冷冻/解冻获得的 PL 相比，通过超声获得的 PL 中 MSC 的 PD 时间（小时）更短（18.9 与 17.4，P<0.01）。BM 有核细胞表达 MSC 标志物，并能够分化为成脂、成骨和成软骨谱系。当 BM-MSCs 和 T 细胞紧密接触共培养时，BM-MSCs 的免疫抑制活性得以维持。细胞核型未显示遗传改变。
通过超声产生 PL 的建议方法可以是 FBS 的安全、高效和快速替代品，而没有 FBS 的潜在风险。
