Journal of blood disorders & transfusion最新文献

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Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials. 用于临床试验的人类内皮祖细胞和内皮细胞的冷冻保存和计数。

Journal of blood disorders & transfusion

Pub Date : 2013-09-20 DOI: 10.4172/2155-9864.1000158

T Bogoslovsky, D Wang, D Maric, L Scattergood-Keepper, M Spatz, S Auh, J Hallenbeck

Background: Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC.

Patients/methods: EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery^™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45^- CD34⁺CD133^+/-VEGFR2^+/- and EC were gated for CD45^-CD146⁺CD34^+/-VEGFR2^+/-.

Results: Cryopreservation for 7 days at -80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at -65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45^- CD34⁺CD133⁺VEGFR2⁺) were enumerated as 0.2 ± 0.06%, late EPC(CD45^-CD146⁺CD34⁺VEGFR²⁺) as 0.6 ± 0.1% and mature EC (CD45^-CD146⁺CD34^-VEGFR2⁺) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at -80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant.

Conclusions: Our data indicate that cryopreservation at -80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.

背景：内皮祖细胞（EPC）是内皮损伤的标志物，可作为介入性临床试验中血管修复的替代标志物。本研究的目的是修改一种分离外周血单核细胞（PBMC）和计数外周血中 EPC 和成熟内皮细胞（EC）的方法，并评估低温保存对 PBMC 活力以及 EPC 和 EC 数量的影响：对健康志愿者用 CPT（细胞制备管）收集的新鲜分离的 PBMC 和用以下方法冷冻保存的 PBMC 中的 EPC 和 EC 进行分析：1）Gibco Recovery™ 细胞培养冷冻培养基；2）定制冷冻培养基。使用 DAPI 检测 PBMC 的活力。对 EPC 进行 CD45- CD34+CD133+/-VEGFR2+/- 分选，对 EC 进行 CD45-CD146+CD34+/-VEGFR2+/- 分选：在-80°C冷冻保存7天后，PBMC的存活率从94 ± 0.5%（新鲜）降至84 ± 4%（定制培养基）和69 ± 8%（Gibco培养基），而在-65°C冷冻保存后，存活率降至60 ± 6%（p- CD34+CD133+VEGFR2+）。2 ± 0.06%，晚期 EPC（CD45-CD146+CD34+VEGFR2+）为活 PBMC 的 0.6 ± 0.1%，成熟 EC（CD45-CD146+CD34-VEGFR2+）为活 PBMC 的 0.8 ± 0.3%。使用 Gibco 和定制冷冻培养基在 -80°C 下冷冻 7 天会减少 EPC 和 EC 的数量，但这种减少并无统计学意义：我们的数据表明，在零下 80 摄氏度条件下冷冻保存 7 天会降低 PBMC 的存活率以及 EC 和 EPC 亚群的数量，但降低幅度不大。这种方法可为EPC亚群和成熟EC的分离和短期冷冻保存提供优化方法，适合多中心试验。

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