The purpose of this study was to explore methods of selectively enriching CD34 + haematopoietic progenitor cells (HPC) in mononuclear cell (MNC) preparations, and to outline a procedure for cryopreservation and thawing of manufactured material. Density gradient centrifugation of umbilical cord blood was achieved using Ficoll-Paque™ media at 1.077 g/mL and 1.065 g/mL densities and Leucosep preparation tubes. Post-process samples were analysed for CD34 + and MNC content. Finally, MNCs were frozen down at a concentration of 8.5 × 10⁶ cells/mL in CryoStor CS10 using an Asymptote VIAFreeze controlled rate freezer at a rate of − 2 °C per minute, then thawed and analysed for viability and recovery. Processing with 1.065 g/mL media selectively depleted non-HPC cell types, producing an approximately fourfold increase in CD34 + frequency (M ± 1SD = 1.4 ± 1.3%, P < 0.01) relative to the pre-process sample (M ± 1SD = 0.4 ± 0.3%), whereas 1.077 g/mL media produced only a twofold enrichment (0.7 ± 0.6, P < 0.01). This was not accompanied by any significant forfeit of CD34 + recovery (79 ± 32% vs. 78 ± 32% respectively; P = 0.87). The MNCs generated by the 1.065 g/mL procedure were of greater purity (96 ± 2%) than in the 1.077 g/mL procedure (80 ± 7%, P < 0.01). Post-thaw, MNC viability was 95 ± 1% and CD34 + viability was 98 ± 1%. Ultra-pure MNCs rich in CD34 + HPCs can be generated with a simple, inexpensive modification to Ficoll-Paque™ media. These products can be easily cryopreserved using a simple controlled rate freezing procedure.

本研究的目的是探索在单核细胞 (MNC) 制剂中选择性富集 CD34 + 造血祖细胞 (HPC) 的方法，并概述制造材料的冷冻保存和解冻程序。使用密度为 1.077 g/mL 和 1.065 g/mL 的 Ficoll-Paque™ 介质以及 Leucosep 制备管实现脐带血的密度梯度离心。分析后处理样品的 CD34 + 和 MNC 含量。最后，使用 Asymptote VIAFreeze 控速冰箱以每分钟 - 2 °C 的速度将 MNC 以 8.5 × 10 6 个细胞/mL 的浓度冷冻在 CryoStor CS10 中，然后解冻并分析活力和恢复^情况。使用 1.065 g/mL 培养基处理选择性地去除非 HPC 细胞类型，相对于预处理 样品 (M ± 1SD = 0.4 ± 0.3%），而 1.077 g/mL 培养基仅产生两倍富集（0.7 ± 0.6，P  < 0.01）。这并不伴随 CD34 + 恢复的任何显着丧失（分别为 79 ± 32% 与 78 ± 32%；P  = 0.87）。1.065 g/mL 程序生成的 MNC 纯度 (96 ± 2%) 高于 1.077 g/mL 程序生成的 MNC (80 ± 7%, P < 0.01 )  。解冻后，MNC 活力为 95 ± 1%，CD34 + 活力为 98 ± 1%。通过对 Ficoll-Paque™ 介质进行简单、廉价的修改，可以生成富含 CD34 + HPC 的超纯 MNC。使用简单的受控速率冷冻程序可以轻松冷冻这些产品。