Innovative Method for Fully Automated, Enzyme-Free Tissue Dissociation and Preparation for Single-Cell Analysis.

IF 5 4区医学 Q3 BIOPHYSICS

Cellular and molecular bioengineering Pub Date : 2025-07-03 eCollection Date: 2025-08-01 DOI:10.1007/s12195-025-00850-5

Sarah Planchak, E Celeste Welch, Benjamin Phelps, Joshua Phelps, Alejandra Hernandez Moyers, Kathryn Whitehead, John Murphy, Nikos Tapinos, Anubhav Tripathi

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引用次数: 0

Abstract

Purpose: Tissue dissociation is a critical but often overlooked step in single-cell analysis, impacting data quality, reproducibility, and biological insights. Conventional enzymatic and mechanical dissociation methods introduce variability, damage cells, and alter transcriptomic profiles, compromising downstream applications. While the initial innovation in electrical dissociation was published, this work introduces expanded characterization, including bulk RNA sequencing, diverse tissue types, and improved flow cytometry.

Methods: Here, we present a fully automated, enzyme-free method that integrates electric field-based dissociation with purification and centrifugation, providing a standardized, scalable alternative. A square wave oscillating electric field at 100 V/cm was used for dissociating tissue samples in 5 minutes or less.

Results: The system rapidly and gently dissociated glioblastoma spheroids and mouse spleen tissue, achieving a 10 × increase in live cell yield compared to automated enzymatic and mechanical dissociation (gentleMACS) and a 96 ± 2% single-cell recovery rate in glioblastoma spheroids. Transcriptomic analysis revealed minimal gene expression changes post-dissociation, with an R² value of 0.997 between conditions, indicating high consistency. Flow cytometry confirmed that key immune cell populations (B, T, NK cells) were preserved, with comparable distributions between manual and electrical dissociation.

Conclusions: By reducing operator variability, improving scalability, and maintaining cellular integrity, this technology offers a robust solution for high-throughput single-cell applications in diagnostics, drug discovery, and precision medicine.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00850-5.

查看原文本刊更多论文

全自动，无酶组织解离和单细胞分析制备的创新方法。

目的：组织分离是单细胞分析中一个关键但经常被忽视的步骤，影响数据质量，可重复性和生物学见解。传统的酶解和机械解离方法引入可变性，损伤细胞，改变转录组谱，影响下游应用。虽然电解离的最初创新已经发表，但这项工作引入了扩展的表征，包括大量RNA测序，不同的组织类型和改进的流式细胞术。方法：在这里，我们提出了一种全自动，无酶的方法，将基于电场的解离与纯化和离心相结合，提供了一种标准化的，可扩展的替代方法。使用100 V/cm的方波振荡电场在5分钟或更短时间内解离组织样品。结果：该系统快速、温和地分离胶质母细胞瘤球状体和小鼠脾组织，与自动酶解和机械解离（gentleMACS）相比，活细胞产量提高10倍，胶质母细胞瘤球状体的单细胞回收率为96±2%。转录组学分析显示，解离后基因表达变化最小，各条件间的R2值为0.997，一致性较高。流式细胞术证实了关键的免疫细胞群（B、T、NK细胞）被保存下来，在手工和电解离之间具有相当的分布。结论：通过减少操作人员的可变性、提高可扩展性和保持细胞完整性，该技术为诊断、药物发现和精准医疗中的高通量单细胞应用提供了强大的解决方案。补充信息：在线版本包含补充资料，可在10.1007/s12195-025-00850-5获得。

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

Cellular and molecular bioengineering 生物-生物物理

CiteScore

5.60

自引率

3.60%

发文量

审稿时长

>12 weeks

期刊介绍： The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas: Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example. Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions. Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress. Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.