Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India.
Homi Bhabha National Institute, Mumbai, India; Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India.
Transplant Cell Ther. 2023 Dec;29(12):777.e1-777.e8. doi: 10.1016/j.jtct.2023.08.032. Epub 2023 Sep 9.
Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34 human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34 stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34 stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r = .03; P = .5). The median CD34 stem cell count in the post-thaw samples was 9.13 × 10/kg (range, .44 to 26.27 × 10/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34 stem cell levels (r = .4; P = .05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.
长期冷冻保存外周血造血干细胞（PBSC）在串联/多次移植或自体造血干细胞移植（HSCT）治疗复发的情况下非常有用。即使在异基因 HSCT 中，如果从供体中收集到过多的干细胞，也可以储存供体淋巴细胞输注数月至数年。冷冻保存是一个精细、复杂和昂贵的过程，常用的冷冻保护剂二甲基亚砜（DMSO）的浓度较高可能对细胞有毒，并在输注过程中对受者产生不良反应。在这项研究中，我们研究了使用 4.35%（最终浓度）二甲亚砜（DMSO）与甲基纤维素和机械冷冻机（-80°C）中无控制速率冷冻长期冷冻保存对 CD34 人类 PBSC 的活力和集落形成能力的影响。对于接受自体 HSCT 的患者，使用含有 4.35%（最终浓度）二甲亚砜（DMSO）的甲基纤维素冷冻保存 PBSC。使用台盼蓝排除法和基于流式细胞术的 7-氨基放线菌素 D（FC-7AAD）方法测定 PBSC 解冻后的活力。使用多色流式细胞术评估解冻后 PBSC 收获样本中 CD34 干细胞和免疫细胞亚群的浓度，并使用集落形成单位（CFU）测定研究解冻后干细胞的克隆形成潜力。使用 Pearson 相关检验将解冻后 CD34 干细胞水平与储存前的 CD34 水平相关联。使用 Bland-Altman 图比较台盼蓝染料排除法和基于流式细胞术的方法的活力结果。我们研究了 26 个 PBSC 收获样本，冷冻保存时间中位数为 6.6 年（范围，3.8 至 11.5 年）。两种方法均显示解冻后 PBSC 的活力>80%，两者之间存在弱一致性（r =.03；P =.5）。解冻后样本中 CD34 干细胞计数的中位数为 9.13×10/kg（范围，0.44 至 26.27×10/kg）。CFU 测定显示解冻后 PBSC 具有良好的增殖和分化潜力，粒细胞巨噬细胞 CFU 与 CD34 干细胞水平之间存在弱相关性（r =.4；P =.05）。两个冷冻保存>8 年的样本活力较低。使用 4.35%（最终浓度）二甲亚砜（DMSO）与甲基纤维素和机械冷冻机（-80°C）中无控制冷冻保存 PBSC 可维持 PBSC 的长期活力长达 8 年。
