Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium
Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium
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DOI：
10.1016/j.cryobiol.2008.08.003
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发表时间：
2008-12-01
期刊：
影响因子：
2.7
通讯作者：
Petrenko, A. Yu.
中科院分区：
文献类型：
--
作者：
Petrenko, Yu. A.;Jones, D. R. E.;Petrenko, A. Yu.
简介：人胎肝（HFL）是造血干/祖细胞（HSC）的宝贵来源，用于治疗各种血液学疾病。本研究描述了在冷冻保护介质中加入蔗糖以降低HFL造血细胞制备物在冷冻保存过程中Me 2SO浓度的效果。方法：将妊娠8-12周的人胎肝（HFL）细胞以1 ℃/min的冷却速率冷冻保存至-80 ℃并储存在液氮中。冷冻保护剂溶液含有2%或5% Me 2SO（v/v），含或不含蔗糖，最终浓度为0.05、0.1、0.2或0.3 M。使用阿利兰测定法测定HFL细胞的代谢活性。为了确定存在的造血祖细胞的数量和存活，用CD 34（FITC）和7-AAD染色细胞，并通过流式细胞术分析。HFL造血干/祖细胞冻存后的集落形成活性进行了评估，在半固体methylcellulose.Results：除了蔗糖的冷冻保护介质产生了显着减少HFL细胞在冻存过程中的损失。用5%Me2SO/0.3M蔗糖混合物冷冻保存的HFL细胞的代谢活性与用5%Me2SO/10%FCS冷冻保存的HFL细胞的代谢活性相当。结论：蔗糖作为HFL细胞冷冻保护液的添加剂，可降低Me 2SO浓度，替代血清，提高冻存效率。(C)2008年爱思唯尔公司All rights reserved.
Introduction: Human fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me2SO concentration during cryopreservation of HFL hematopoietic cell preparations.Methods: Human fetal liver (HFL) cells of 8-12 weeks of gestation were cryopreserved with a cooling rate of 1 degrees C/min down to -80 degrees C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me2SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoictic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.Results: The addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me2SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me2SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34(+) cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.Conclusion: The inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me2SO, replacing serum and increasing the efficiency of cryopreservation. (C) 2008 Elsevier Inc. All rights reserved.