Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells
Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells
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DOI：
10.1634/stemcells.2004-0135
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发表时间：
2005-05-01
期刊：
影响因子：
5.2
通讯作者：
Schwarz, J
中科院分区：
文献类型：
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作者：
Milosevic, J;Storch, A;Schwarz, J
干细胞研究为开发针对毁灭性疾病的新医学疗法提供了独特的机会，并为探索生物学基本问题提供了新的途径。建立一种有效的神经前体细胞（NPC）冷冻方案对于细胞治疗的进展非常重要。我们使用基于荧光激活细胞分选仪的细胞死亡/存活分析和增殖标记物（增殖细胞核抗原）和促存活蛋白（Bcl-2）的Western印迹分析，研究各种冷冻保护剂对胎鼠前脑NPC的影响。以速率控制的方式在-70° C或液氮中冷冻并在5天后解冻的神经球在解冻后24小时测量保留60%-70%的活力。然而，解冻后1周，活力下降至50%-60%。使用克隆球形成试验，我们发现，冷冻NPC的回收率约为26%，并没有显着差异二甲亚砜（DMSO）和甘油补充样品。在冷冻过程中或解冻后第一周内应用半胱天冬酶抑制剂zVAD-fetamine可保护解冻后冷冻保存的神经球，但在冷冻过程中则不然，这表明细胞凋亡限制了NPC的恢复。在冷冻保存前酶促分离的细胞中，细胞存活率没有降低。当单独使用10%DMSO或与10%胎牛血清（FCS）组合使用时，实现了NPC的最佳保护。然而，单独使用10%甘油同样有效。使用这些协议，NPC保留其多能性，并分化成神经胶质细胞（GFAP阳性）和神经元细胞（Tuj 1阳性）。在5%和10%DMSO、10%DMSO +10%FCS和10%甘油中的Tuj 1阳性细胞百分比保持在与冷冻前相同的水平，并且在5%-7%之间变化。我们得出结论，冷冻保存（在-70° C下长达1个月，在液氮中长达1年）不会显著改变小鼠神经前体细胞的增殖率和多能性。
Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70° C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, l week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)- and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70° C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.