Zhu Fenlu, Heditke Sarah, Kurtzberg Joanne, Waters-Pick Barbara, Hari Parameswaran, Margolis David A, Keever-Taylor Carolyn A
Department of Medicine, Hematology & Oncology Division, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Froedtert Hospital, Milwaukee, Wisconsin, USA.
Cytotherapy. 2015 Dec;17(12):1813-9. doi: 10.1016/j.jcyt.2015.08.007. Epub 2015 Oct 9.
Removing DMSO post-thaw results in: reduced infusion reactions, improved recovery and stability of viable CD34+ cells. Validated methods use 5%-8.3% Dextran 40 with 2.5%-4.2% HSA for this purpose. Recent shortages of clinical grade Dextran require identification of suitable alternatives.
PBPC were used to compare a standard 2X wash medium of 5 parts 10% Dextran 40 in saline (DEX) with 1 part 25% HSA (8.3% DEX/ 4.2% HSA) with Hydroxyethyl Starch (HES)-based solutions. Cells in replicate bags were diluted with an equal volume of wash solution, equilibrated 5 minutes, the bag filled with wash medium, pelleted and the supernatant expressed. Bags were restored to the frozen volume in wash medium and tested by single platform flow cytometry and CFU. Total viability, viable TNC, MNC, and CD34+ cell recovery, and CD34+ cell viability were compared immediately post-thaw and after 90 minutes.
5.2% HES/4.2% HSA did not differ from our standard in CD34 recovery or viability. Due to concerns that high concentrations of HES could affect renal function we tested 0.6% HES/2.5% HSA resulting in significantly poorer CD34 recovery and viability. Results improved using 2.4% HES/4.2% HSA and when 0.6% HES/4.2%HSA was used no significant differences were seen. CFU assays confirmed no differences between the standard dextran arm and HES at 2.4% or 0.6% so long as HSA was at 4.2%.
We conclude that HES from 0.6% to 5.2% with 4.2% HSA is a suitable substitute for Dextran 40 as a reconstitution/washing medium for PBPC products.
解冻后去除二甲基亚砜（DMSO）可带来以下结果：减少输注反应，提高存活的CD34+细胞的回收率和稳定性。为此，经过验证的方法使用5%-8.3%的右旋糖酐40与2.5%-4.2%的人血清白蛋白（HSA）。近期临床级右旋糖酐的短缺需要确定合适的替代品。
使用外周血祖细胞（PBPC）将标准的2倍洗涤培养基（5份10%的右旋糖酐40生理盐水溶液（DEX）与1份25%的HSA（8.3% DEX/4.2% HSA））与基于羟乙基淀粉（HES）的溶液进行比较。将复制品袋中的细胞用等体积的洗涤溶液稀释，平衡5分钟，向袋中加入洗涤培养基，离心沉淀并弃去上清液。将袋子恢复到洗涤培养基中的冷冻体积，通过单平台流式细胞术和集落形成单位（CFU）进行检测。在解冻后立即和90分钟后比较总活力、存活的有核细胞（TNC）、单核细胞（MNC）和CD34+细胞回收率以及CD34+细胞活力。
5.2% HES/4.2% HSA在CD34回收率或活力方面与我们的标准无差异。由于担心高浓度的HES会影响肾功能，我们测试了0.6% HES/2.5% HSA，结果导致CD34回收率和活力明显较差。使用2.4% HES/4.2% HSA时结果有所改善，当使用0.6% HES/4.2% HSA时未观察到显著差异。CFU检测证实，只要HSA为4.2%，标准右旋糖酐组与2.4%或0.6%的HES组之间无差异。
我们得出结论，0.6%至5.2%的HES与4.2%的HSA是作为PBPC产品的重构/洗涤培养基替代右旋糖酐40的合适选择。
