Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates
Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates
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DOI：
10.1080/0953710031000092811
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发表时间：
2003-05-01
期刊：
影响因子：
3.3
通讯作者：
Meryman, HT
中科院分区：
文献类型：
--
作者：
Arnaud, F;Kapnik, E;Meryman, HT
据推测，除了冷冻损伤外，在去除冷冻保护剂过程中发生的细胞挤压可能会对血小板造成一些损伤。本研究考察了通过中空纤维（HF）过滤器进行液体交换来去除二甲基亚砜（DMSO），以此作为离心法的替代方法。含或不含细胞悬液的DMSO溶液都通过过滤器一次。通过改变HF过滤器内外腔室的流速来确定DMSO卸载过程中的最佳交换条件。最初，将不含血小板的5%DMSO缓冲溶液泵入纤维中，并与磷酸盐缓冲盐水（PBS）进行交换。通过渗透压测定法测定残留的DMSO。DMSO跨膜交换与流速有关，也受HF纤维化学性质的影响。任何以合理流速通过纤维的方案单次通过都不能去除超过95%的DMSO。使用聚辛烷纤维，内部流速约为20毫升/分钟，外部流速为100毫升/分钟时可达到最佳方案。随后，使用离心法洗涤DMSO中的冻融血小板浓缩物，并与HF过滤法进行比较。通过流式细胞术、细胞计数、形态学和渗透压应激试验检测血小板质量。过滤和离心洗涤技术都产生了相当的形态学评分和盘状细胞数量。当加入减少血小板活化的试剂时，两种技术洗涤后的血小板质量都得到了改善。在使用活化抑制剂时，HF过滤法的血小板渗透压反应低于离心法，这归因于抑制剂的剩余量。所有其他测试参数都相似。两种技术的CD62P表达相当，而且与最初预期相反，离心法激活血小板并不比过滤法更严重。总之，两种技术洗涤后的血小板质量相当，但中空纤维过滤去除冷冻保护剂比离心法更快。
It has been hypothesized that, in addition to freezing injury, some damage to platelets may result from the cell packing that occurs during removal of the cryoprotectant. This study examined DMSO removal by fluid exchange across hollow-fiber (HF) filters as an alternative to centrifugation. The DMSO solution with or without cell suspension was passed once through the filter. The optimum exchange during unloading of DMSO was determined by varying the flow rates in the external and internal compartments of the HF filter. Initially, buffered solutions of a 5% DMSO solution in the absence of platelets were pumped into the fibers and exchanged against PBS. The residual DMSO was determined by osmometry. The exchange of DMSO across the membrane was flow dependent and also influenced by the chemical nature of the HF fibers. No protocol using a reasonable rate flow through the fibers removed more than 95% of the DMSO in a single pass. The optimum protocol was achieved with polysynthane fibers with an internal flow rate of approximately 20 ml/min and an external flow rate of 100 ml/min. Subsequently, frozen/thawed platelet concentrates in DMSO were washed using centrifugation and compared to the HF filtration method. Platelet quality was assayed by flowcytometry, cell count, morphology and osmotic stress test. Both filtration and centrifugal washing techniques resulted in comparable morphological scores and numbers of discoid cells. When agents reducing platelet activation were added, platelet quality was improved after washing by either technique. The lower platelet osmotic response with HF filtration than with centrifugation while using activation inhibitors was attributed to the remaining amount of the inhibitors. All other parameters tested were similar. The expression of CD62P was equivalent with both techniques, and centrifugation did not activate platelets more than filtration contrary to what was originally anticipated. In conclusion, platelet quality was comparable after washing by either technique but hollow fiber filtration does remove cryoprotectant more rapidly than does centrifugation.