Activation of mitochondrial-associated apoptosis contributes to cryopreservation failure
Activation of mitochondrial-associated apoptosis contributes to cryopreservation failure
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DOI：
10.1089/cpt.2007.9990
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发表时间：
2007-09-01
影响因子：
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通讯作者：
Baust, John G.
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文献类型：
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作者：
Baust, John M.;Vogel, Martin J.;Baust, John G.
对于许多细胞系统来说，冷冻保存(CP)后解冻后的存活率仍然不是最理想的。最近对延迟性细胞死亡(DOCD)的关注为这一观察到的失败提供了解释。DOCD激活的一个潜在途径涉及内在的线粒体-凋亡途径。具体地说，应激源可以破坏与线粒体和相关的Bcl-2蛋白家族相关的促/抗凋亡蛋白平衡。我们推测，依赖CP的促/抗凋亡比率的破坏(具体而言，bax：bclX-L)有助于DOCD的激活和进展。在这项研究中，人皮肤成纤维细胞在不同的冷冻介质(介质+5%DMSO或CryoStor(TM)CS5)中冷冻保存，在VC中心点min(-1)至-80℃冷冻，并保存在LN2中。细胞保存后，快速解冻，接种于培养液中。每天使用代谢指示剂(AlamarBlue)和核酸探针(SytoDye)评估生存能力。分别于解冻后0、6、12、24 h分离细胞总蛋白，Western blotting检测bclX-L和bax的表达。结果：(1)在解冻24 h后，使用CryoStor(TM)CS5的细胞存活率比添加5%DMSO的细胞存活率增加了61%和37%。(2)超低温保存后，细胞的促/抗凋亡比例被破坏，在恢复期向促死亡信号转移。(3)使用CryoStor(TM)CS5后，促/抗细胞凋亡率的变化比在5%二甲基亚砜(5%DMSO)中保存的细胞减少了几倍。总而言之，对CP后线粒体蛋白水平的检查揭示了不同CP方案之间不同的时间分布。这项研究表明，线粒体相关的凋亡通路的激活在CP诱导的DOCD的执行中起着不可或缺的作用。这项研究进一步表明，控制细胞凋亡，特别是线粒体相关的死亡，可能有助于进一步提高CP后的细胞存活率。
Post-thaw viability following cryopreservation (CP) remains suboptimal for many cell systems. Recent focus on delayed-onset cell death (DOCD) is providing an explanation for this observed failure. One potential avenue for the activation of DOCD involves the intrinsic mitochondrial-apoptotic pathway. Specifically, stressors can disrupt the pro-/anti-apoptotic protein balance associated with the mitochondria and the related Bcl-2 protein family. We hypothesized that CP-dependent disruption of the pro-/anti-apoptotic ratio (specifically, Bax: BCl-X-L contributes to the activation and progression of DOCD. In this study, human dermal fibroblasts were cryopreserved in various freeze media (media +5% DMSO or CryoStor (TM) CS5), frozen at VC center dot min(-1) to -80 degrees C and stored in LN2. Following storage, cells were rapidly thawed and plated in culture media. Viability was assessed daily using a metabolic indicator (alamarBlue) and a nucleic acid probe (SytoDye). Total cellular protein was isolated from samples at 0, 6, 12, and 24 h post-thaw, and BCl-X-L and Bax levels were analyzed via Western blotting. Results: (1) utilization of CryoStor (TM) CS5 yielded an increase in cell survival over that of media +5% DMSO (61% vs. 37%, 24 h post-thaw). (2) Cryopreservation resulted in the disruption of the pro-/anti-apoptotic ratio, shifting towards prodeath signaling during the recovery period. (3) Utilization of CryoStor (TM) CS5 decreased the shift in the pro-/antiapoptotic ratio compared with storage in Media +5% DMSO (severalfold). In conclusion, examination of mitochondrial protein levels following CP revealed distinct temporal profiles between differing CP protocols. This study demonstrates that activation of the mitochondrial-associated apoptotic pathway plays an integral role in the execution of CP-induced DOCD. This study further illustrates that the control of apoptotic cell death, particularly mitochondrial associated, may facilitate further enhancement in cell survival following CP.