Ma Jinxia, Shao Lipei, Fuksenko Tatyana, Liu Hui, Jiang Chunjie, Cai Yihua, Kim Yong Soo, Martin Kathryn, Moses Larry, Zhang Nan, Dinh Anh, Somerville Robert P, Stroncek David F, Jin Ping
Center for Cellular Engineering, Department of Transfusion Medicine and Center for Cellular Engineering, NIH Clinical Center, Bethesda, MD 20892, USA.
Center for Cellular Engineering, Department of Transfusion Medicine and Center for Cellular Engineering, NIH Clinical Center, Bethesda, MD 20892, USA.
Immunobiology. 2025 Aug 29;230(6):153112. doi: 10.1016/j.imbio.2025.153112.
Hematopoietic progenitor cells (HPCs) and mononuclear cells (MNCs) are critical components of cell-based therapies, including bone marrow transplantation and regenerative treatments. Evaluation of the characteristics of these products during collection, storage, and transport is essential for maintaining cell viability and functionality. In this study, we evaluated the functional and molecular stability of samples collected for the evaluation of fresh HPC and MNC products. The samples stored at 4 °C for up to 4 days and were evaluated using white blood cell (WBC) counts, flow cytometry, and bulk RNA sequencing (RNA-seq) across five time points.
HPC samples from seven products (June-December 2022) and MNC samples from six products (October 2022-August 2023) were analyzed on days 0 through 4 after collection. WBC counts were measured, and viability was assessed using 7-AAD staining and flow cytometry. HPC samples were stained with antibodies against CD34, CD3, CD19, CD56, CD14, CD16, CD15, and CD45, while MNC samples were stained with antibodies directed to CD3, CD4, CD8, CD19, CD56, CD14, CD16, CD15, and CD45. Total RNA was isolated from each sample and subjected to bulk RNA-seq to assess transcriptomic changes during storage.
While WBC counts varied between products, no significant differences were observed across time points within individual products. Flow cytometry markers remained relatively stable over time in both HPC and MNC samples, although greater variability was observed in HPCs. A modest decrease in lymphocyte percentages was noted at later time points, primarily driven by a reduction in CD3+ cells; however, these changes were not statistically significant. Cell viability declined significantly over time within individual products and showed inter-product variability. RNA-seq analysis revealed stable gene expression profiles in MNC samples across all time points. In contrast, HPC samples exhibited notable transcriptomic changes as early as day 1 of storage at 4 °C, indicating greater molecular instability.
WBC counts and flow cytometry markers remain stable for up to 3 days in samples collected from fresh HPC and MNC products when stored at 4 °C, although cell viability progressively declines. However, RNA-seq data reveal early transcriptomic changes in HPC samples, suggesting that immediate evaluation of these samples is critical to preserve their molecular integrity and functionality. These findings support the feasibility of delayed phenotypic analysis but emphasize the need for prompt molecular assays in HPC-based applications.
造血祖细胞（HPCs）和单核细胞（MNCs）是细胞疗法的关键组成部分，包括骨髓移植和再生治疗。在采集、储存和运输过程中评估这些产品的特性对于维持细胞活力和功能至关重要。在本研究中，我们评估了采集用于新鲜HPC和MNC产品评估的样本的功能和分子稳定性。样本在4℃下储存长达4天，并在五个时间点使用白细胞（WBC）计数、流式细胞术和批量RNA测序（RNA-seq）进行评估。
对2022年6月至12月的7种产品的HPC样本和2022年10月至2023年8月的6种产品的MNC样本在采集后的第0天至第4天进行分析。测量WBC计数，并使用7-AAD染色和流式细胞术评估活力。HPC样本用抗CD34、CD3、CD19、CD56、CD14、CD16、CD15和CD45的抗体染色，而MNC样本用针对CD3、CD4、CD8、CD19、CD56、CD14、CD16、CD15和CD45的抗体染色。从每个样本中分离总RNA，并进行批量RNA-seq以评估储存期间的转录组变化。
虽然不同产品之间的WBC计数有所不同，但在单个产品的各个时间点未观察到显著差异。在HPC和MNC样本中，流式细胞术标志物随时间保持相对稳定，尽管在HPC中观察到更大的变异性。在后期时间点，淋巴细胞百分比略有下降，主要由CD3+细胞减少驱动；然而，这些变化没有统计学意义。在单个产品中，细胞活力随时间显著下降，并表现出产品间的变异性。RNA-seq分析显示MNC样本在所有时间点的基因表达谱稳定。相比之下，HPC样本在4℃储存的第1天就表现出显著的转录组变化，表明分子不稳定性更大。
从新鲜HPC和MNC产品采集的样本在4℃储存时，WBC计数和流式细胞术标志物在长达3天内保持稳定，尽管细胞活力逐渐下降。然而，RNA-seq数据显示HPC样本早期有转录组变化，这表明对这些样本进行即时评估对于保持其分子完整性和功能至关重要。这些发现支持延迟表型分析的可行性，但强调在基于HPC的应用中需要及时进行分子检测。
