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优化的冷冻人肾组织组学处理方案。

Optimized protocol for the multiomics processing of cryopreserved human kidney tissue.

机构信息

III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Hamburg Center for Kidney Health, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

出版信息

Am J Physiol Renal Physiol. 2024 Nov 1;327(5):F822-F844. doi: 10.1152/ajprenal.00404.2023. Epub 2024 Oct 3.


DOI:10.1152/ajprenal.00404.2023
PMID:39361723
Abstract

Biobanking of tissue from clinically obtained kidney biopsies for later analysis with multiomic approaches, such as single-cell technologies, proteomics, metabolomics, and the different types of imaging, is an inevitable step to overcome the need of disease model systems and toward translational medicine. Hence, collection protocols that ensure integration into daily clinical routines by the usage of preservation media that do not require liquid nitrogen but instantly preserve kidney tissue for both clinical and scientific analyses are necessary. Thus, we modified a robust single-nucleus dissociation protocol for kidney tissue stored snap-frozen or in the preservation media RNAlater and CellCover. Using at first porcine kidney tissue as a surrogate for human kidney tissue, we conducted single-nucleus RNA sequencing with the widely recognized Chromium 10X Genomics platform. The resulting datasets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques such as proteomics, metabolomics, and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines, the preservation medium RNAlater facilitates the collection of highly preserved human kidney biopsies and enables further analysis with cutting-edge techniques like single-nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap-frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening up new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases. In this study, we addressed challenges in integrating clinically obtained kidney biopsies into everyday clinical routines. Using porcine kidneys, we evaluated preservation media (RNAlater and CellCover) versus snap freezing for multi-omics processing. Our analyses highlighted RNAlater's suitability for single-nucleus RNA sequencing, proteome analysis and histopathological evaluation. Only metabolomics are currently restricted to snap-frozen biopsies. Our research established a cryopreservation protocol that facilitates tissue biobanking for advancing precision medicine in nephrology.

摘要

从临床上获得的肾活检组织中进行生物银行储存,以便以后使用多组学方法(如单细胞技术、蛋白质组学、代谢组学和不同类型的成像)进行分析,是克服疾病模型系统的需要并迈向转化医学的必然步骤。因此,需要收集方案,通过使用不需要液氮但能立即保存肾组织用于临床和科学分析的保存介质,确保将其纳入日常临床常规。因此,我们修改了一个稳健的用于储存 snap 冷冻或在保存介质 RNAlater 和 CellCover 中的肾组织的单细胞核分离方案。我们首先使用猪肾组织作为人肾组织的替代品,使用广泛认可的 10X Genomics Chromium 平台进行单细胞 RNA 测序。然后,分析来自每种储存条件的数据集,以鉴定转录组谱中任何潜在的变化。此外,我们评估了保存介质是否适合其他分析技术,如蛋白质组学、代谢组学以及组织架构的保存,以便进行包括免疫荧光染色在内的组织病理学检查。在这项研究中,我们表明在日常临床常规中,保存介质 RNAlater 促进了高度保存的人类肾活检的收集,并使单细胞 RNA 测序、蛋白质组学和组织病理学评估等前沿技术得以进一步分析。目前只有代谢组分析受到 snap 冷冻组织的限制。这项工作将有助于建立具有明确肾脏疾病队列的组织生物银行,对其进行深入的分子特征分析,为预防、早期识别和靶向治疗肾脏疾病的独特细胞、途径和生物标志物的识别开辟新的视野。在这项研究中,我们解决了将临床上获得的肾活检组织纳入日常临床常规的挑战。使用猪肾,我们评估了保存介质(RNAlater 和 CellCover)与 snap 冷冻在多组学处理方面的比较。我们的分析强调了 RNAlater 用于单细胞 RNA 测序、蛋白质组分析和组织病理学评估的适用性。目前只有代谢组学受到 snap 冷冻活检的限制。我们的研究建立了一种冷冻保存方案,为肾脏病学中的精准医学提供了组织生物银行。

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引用本文的文献

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[2]
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