Advanced Cell Therapy Core, Sidra Medicine, Doha, Qatar.
Pediatric Oncology and Hematology Division, Sidra Medicine, Doha, Qatar.
Transfusion. 2023 May;63(5):1050-1059. doi: 10.1111/trf.17338. Epub 2023 Apr 10.
Cryopreservation and thawing protocols represent key factors for the efficacy of cellular therapy products, such as hematopoietic stem cells (HSCs). While the HSC cryopreservation has already been standardized, the thawing procedures have been poorly studied. This study aimed to evaluate the thawing and washing protocol of cord blood (CB) derived HSCs or the HPC(CB), by selecting the optimal thawing solution and determining CD34+ cells' stability over time.
Seven cryopreserved CB products were thawed, washed, and resuspended in three different solutions (10% Dextran40 in NaCl equally prepared with 5% human albumin; 5% human albumin in PBS/EDTA; and normal saline) and stored at 4°C (±2°C). Mononuclear cell (MNC) count, CD45+/CD34+ cell enumeration, and cell viability were tested at 0, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. The protocol with the selected solution was further validated on additional 10 CB samples. The above parameters and the colony-forming unit (CFU) assay were analyzed at time points 0, 2, 4, 6, and 8 h.
The results showed that the 5% human albumin was the most suitable thawing solution. MNCs were stable up to 4 h (p = 0.009), viable CD45+ cells were unstable even at 2 h (p = 0.013), and viable CD34+ cells were stable until 6 h (p = 0.019). The CFU assay proved the proliferative potential up to 8 h, although significantly decreased after 4 h (p = 0.013), and correlated with the viable CD34+ cell counts. We demonstrated that the post-thawed and washed HPC(CB) using 5% human albumin is stable for up to 4 h.
细胞治疗产品（如造血干细胞 [HSCs]）的疗效与冷冻保存和复温方案密切相关。虽然 HSC 的冷冻保存已经标准化，但复温程序的研究还不够充分。本研究旨在通过选择最佳复温溶液并确定 CD34+细胞随时间的稳定性，来评估脐带血（CB）来源的 HSCs 或 HPC（CB）的复温及洗涤方案。
对 7 个冷冻保存的 CB 产品进行复温、洗涤和重悬于三种不同的溶液（用等渗盐水等量制备的 10%葡聚糖 40 和 5%人血白蛋白；PBS/EDTA 中的 5%人血白蛋白；生理盐水）中，并储存在 4°C（±2°C）。在 0、1、2、4、6、8、12、24、36 和 48 小时时检测单核细胞（MNC）计数、CD45+/CD34+细胞计数和细胞活力。选择的溶液后，对另外 10 个 CB 样本进行了进一步验证。在 0、2、4、6 和 8 小时时分析上述参数和集落形成单位（CFU）检测。
结果表明，5%人血白蛋白是最合适的复温溶液。MNC 稳定至 4 小时（p=0.009），活 CD45+细胞在 2 小时时就不稳定（p=0.013），活 CD34+细胞在 6 小时时稳定（p=0.019）。CFU 检测证明了增殖潜力可长达 8 小时，但在 4 小时后显著下降（p=0.013），与活 CD34+细胞计数相关。我们证明，使用 5%人血白蛋白复温并洗涤后的 HPC（CB）在 4 小时内保持稳定。
