Kozlowska-Skrzypczak M, Kubiak A, Bembnista E, Matuszak P, Komarnicki M
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
Transplant Proc. 2014 Oct;46(8):2535-8. doi: 10.1016/j.transproceed.2014.08.023.
Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system.
Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (-160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 10(8)/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland).
The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99.1% living cells.
用于自体移植的造血干细胞的冷冻保存是库存储过程中的关键环节。尽管冷冻保存技术广为人知，但仍需改进。本研究旨在优化冷冻保存，以改善移植材料中造血干细胞的定量和定性参数。我们利用现有机会，在封闭系统中提供处理后的造血干细胞移植的最佳定量和定性参数。
本报告基于通过白细胞分离术从淋巴增殖性疾病患者中采集的248份造血干细胞产品。材料在程序降温冷冻机中冷冻，并储存在液氮（-160°C）气相中的容器中。冷冻保护剂培养基的成分进行了改良。冷冻时，192个样本使用含有20%二甲基亚砜（DMSO）和强化RPMI 1640的冷冻保护剂培养基。对于56个样本，我们使用白细胞分离术中采集的自体血浆中的20% DMSO。造血干细胞产品与冷冻保护剂培养基按1:1比例混合。最终核细胞数量不超过2×10(8)/mL。样本解冻后进行分析。使用台盼蓝孵育后通过显微镜技术评估核细胞活力，使用7-氨基放线菌素D通过流式细胞术评估CD34+细胞活力。使用Statistica程序（StatSoft，波兰克拉科夫）进行统计分析。
结果表明，使用自体血浆与核细胞和CD34+细胞的更高活力相关。此外，使用RPMI 1640和自体血浆冷冻的组之间，核细胞和CD34+细胞活力的统计分析存在显著差异（P <.05）。为评估CD34+细胞活力，使用RPMI 1640冷冻的细胞结果显示活细胞跨度较大，为16.4%至99.1%。
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