Dynamic effects of short-term storage temperature and duration on the transcriptome and functional pathways of umbilical cord blood-derived NK cells

IF 1.7 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods Pub Date : 2026-09-01 Epub Date: 2026-08-29 DOI:10.1016/j.jim.2026.114110

Jing Xu, Yanqin Wang, Dongmei Yang, Zehua Zhang, Li Zhang, Shanshan Xu, Jing Dong, Xuesong Gao, Chenyu Liu

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引用次数: 0

Abstract

Objective

This study aims to elucidate the dynamic impact of different storage conditions (temperature and time) on the function of natural killer (NK) cells derived from umbilical cord blood (UCB) at the transcriptome level, providing a theoretical basis for optimizing the standardized post-collection processing protocol of UCB in clinical settings.

Methods

Four healthy full-term UCB samples were collected and assigned to a fresh control group (0H) and experimental groups stored at 4 °C (4C) or 25 °C (RT) for 24 h (24H) and 72 h (72H). Umbilical cord blood mononuclear cells (CBMCs) were isolated and expanded in vitro to derive NK cells. Using RNA sequencing (RNA-seq) technology, combined with principal component analysis (PCA), screening of differentially expressed genes (DEGs), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the effects of storage conditions on the transcriptome of NK cells were comprehensively analyzed.

Results

The results indicated that storage time was the primary factor causing transcriptomic differences. Compared to the 0H group, storage for 24 h induced a limited number of Differentially Expressed Genes (DEGs) (only 11 in the 24H-RT group), indicating overall transcriptome stability. In contrast, storage for 72 h triggered profound transcriptomic reprogramming, with DEGs significantly enriched in immune activation-related pathways. Specifically, at the 72-h time point, storage at 4 °C compared to 25 °C prevented the downregulation of key metabolic pathways such as ‘response to decreased oxygen levels’ and ‘regulation of leukocyte cell-cell adhesion’.

Conclusion

CBMC-derived NK cells can tolerate up to 24 h of ex vivo storage while maintaining transcriptome and core functional stability. However, extending the ex vivo storage period to 72 h leads to significant reprogramming in the expression of immune and metabolism-related genes. Notably, the degree of metabolic suppression under refrigerated conditions at 4 °C was less pronounced than that at 25 °C room temperature storage. This study recommends that the ex vivo storage time of UCB should ideally be limited to within 24 h, with room temperature storage being a viable option. If prolonged storage is necessary, refrigeration at 4 °C is advised.

查看原文本刊更多论文

短期储存温度和时间对脐血NK细胞转录组和功能通路的动态影响。

目的：本研究旨在阐明不同储存条件（温度和时间）对脐带血自然杀伤细胞（NK）转录组水平功能的动态影响，为优化临床脐带血标准化采集后处理方案提供理论依据。方法：收集4例健康足月UCB标本，分为新鲜对照组（0H）和试验组(4 °C （4C）和25 °C （RT）保存24 h （24H）和72 h （72H）。分离脐血单个核细胞（CBMCs），体外扩增获得NK细胞。采用RNA测序（RNA-seq）技术，结合主成分分析（PCA）、差异表达基因筛选（DEGs）、基因本体（GO）、京都基因与基因组百科全书（KEGG）途径富集分析，全面分析了储存条件对NK细胞转录组的影响。结果：结果表明，贮藏时间是造成转录组差异的主要因素。与0H组相比，24 h的储存诱导了有限数量的差异表达基因（deg）（24H-RT组只有11个），表明整体转录组稳定。相比之下，储存72 h引发了深刻的转录组重编程，其中DEGs在免疫激活相关途径中显著富集。具体来说，在72小时的时间点，与25 °C相比，4 °C的储存阻止了关键代谢途径的下调，如“对低氧水平的反应”和“白细胞细胞-细胞粘附的调节”。结论：cbmc衍生的NK细胞可以耐受24 h的体外储存，同时保持转录组和核心功能的稳定性。然而，将离体贮藏期延长至72 h会导致免疫和代谢相关基因的表达发生显著的重编程。值得注意的是，4 °C冷藏条件下的代谢抑制程度不如25 °C室温储存时明显。本研究建议UCB的离体保存时间最好限制在24 h以内，室温保存是可行的选择。如果需要长期储存，建议在4 °C冷藏。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of immunological methods 医学-免疫学

CiteScore

4.10

自引率

0.00%

发文量

120

审稿时长

3 months

期刊介绍： The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.