Introduction

While single-cell analysis technology has flourished, obtaining single cells from complex tissues continues to be a challenge. Current methods require multiple steps and several hours of processing. This study investigates chemical and mechanical methods for clinically relevant preparation of single-cell suspension from frozen biopsy cores of complex tissues. The developed protocol can be completed in 15 min.

Methods

Frozen bovine liver biopsy cores were normalized by weight, dimension, and calculated cellular composition. Various chemical reagents were tested for their capability to dissociate the tissue via confocal microscopy, hemocytometry and quantitative flow cytometry. Images were processed using ImageJ. Quantitative flow cytometry with gating analysis was also used for the analysis of dissociation. Physical modeling simulations were conducted in COMSOL Multiphysics.

Results

A rapid method for tissue dissociation was developed for single-cell analysis techniques. The results of this study show that a combination of 1% type-1 collagenase and pronase or hyaluronidase in 100 U/µL HBSS solution is the most effective at dissociating 2.5 mm thawed bovine liver biopsy cores in 15 min, with dissociation efficiency of 37-42% and viability >90% as verified using live MDA-MB-231 cancer cells. Cellular dissociation is significantly improved by adding a controlled mechanical force during the chemical process, to dissociate 93 ± 8% of the entire tissue into single cells.

Conclusions

Understanding cellular dissociation in ex vivo tissues is essential to the development of clinically relevant dissociation workflows. Controlled mechanical force in combination with chemical treatment produces high quality tissue dissociation. This research is relevant to the understanding and assessment of tissue dissociation and the establishment of an automated preparatory workflow for single cell diagnostics.

中文翻译：

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用于单细胞分析的临床相关化学机械组织解离工作流程的优化

介绍

虽然单细胞分析技术蓬勃发展，但从复杂组织中获取单细胞仍然是一个挑战。当前的方法需要多个步骤和几个小时的处理。本研究调查了从复杂组织的冷冻活检核心制备单细胞悬液的临床相关制备方法的化学和机械方法。开发的协议可在 15 分钟内完成。

方法

冷冻牛肝活检核心按重量、尺寸和计算的细胞组成进行标准化。通过共聚焦显微镜、血细胞计数和定量流式细胞术测试了各种化学试剂分离组织的能力。使用 ImageJ 处理图像。具有门控分析的定量流式细胞术也用于解离分析。物理建模仿真在 COMSOL Multiphysics 中进行。

结果

为单细胞分析技术开发了一种快速的组织解离方法。本研究结果表明，100 U/µL HBSS 溶液中的 1% 1 型胶原酶和链霉蛋白酶或透明质酸酶的组合在 15 分钟内解离 2.5 mm 解冻的牛肝活检核心最有效，解离效率为 37-使用活的 MDA-MB-231 癌细胞证实为 42% 和 >90% 的存活率。通过在化学过程中添加受控机械力可显着改善细胞解离，将整个组织的93 ± 8% 解离为单个细胞。

结论

了解离体组织中的细胞解离对于开发临床相关解离工作流程至关重要。受控机械力与化学处理相结合，可产生高质量的组织解离。这项研究与组织解离的理解和评估以及单细胞诊断的自动化准备工作流程的建立有关。