高效和稳定提取原代成年小鼠心脏成纤维细胞的新方法

A new method for extracting adult mouse cardiac fibroblasts more efficiently and stably

摘要

目的：心脏成纤维细胞（cardiac fibroblasts，CFs）在心肌重塑和纤维化进程中发挥核心作用，其高效获取是开展相关机制研究的先决条件。目前，提取原代成年小鼠 CFs 的方法存在耗时长、得率低、活性差等问题，本研究旨在通过优化酶解体系与机械解离参数的协同作用，建立快速、高产、稳定的成年小鼠 CFs 分离方案。方法：借助全自动组织解离器（gentleMACS  ^®  Octo Dissociator with Heaters），选择不同种类及浓度的胶原酶、胰蛋白酶和核酸酶作为酶解体系，提取成年小鼠 CFs ，探索更优化的提取条件（优化组），并与商品化多组织解离试剂盒2（Multi TissueDissociation Kit 2，试剂盒组）的提取效果进行比较。采用高内涵成像分析系统统计贴壁 72 h 后的每视野细胞数量，检测细胞得率；用免疫荧光法标记波形蛋白（vimentin），鉴定 CFs 的纯度；用转化生长因子 β1（transforming growthfactor β1 ，TGF-β1）刺激 CFs，判断转分化活性。结果：胶原酶、胰蛋白酶和核酸酶三者缺少任意一种都将延长细胞提取时间，降低细胞得率。优化组比商品化试剂盒组的消化时长缩短 32.2 min ， CFs 得率显著提高。在 TGF-β1 处理后，CFs 的增殖能力增强，α-平滑肌肌动蛋白、I 型胶原和纤连蛋白的表达量上调。结论：本研究依托组织解离器，优化了一种基于胶原酶、胰蛋白酶和核酸酶的混合酶消化法，能够高效、稳定地提取具有正常分化潜能的成年小鼠 CFs。

Abstract

Objective：Cardiac fibroblasts（CFs）play a central role in myocardial remodeling and fi- brosis.Efficient isolation of CFs is a prerequisite for investigating related mechanisms.However，current methods for isolating primary adult mouse CFs suffer from prolonged processing time，low yield，and poor viability.This study aims to establish a rapid，high-yield，and stable isolation protocol for adult mouse CFs by optimizing the synergistic effect of enzymatic digestion and mechanical dissociation parameters. Methods：Using the gentleMACS  ^®  Octo Dissociator with Heaters，we selected different types and con- centrations of collagenase，trypsin，and nuclease as the enzymatic digestion system for CFs extraction. We explored the optimal extraction conditions and compared the results with the commercial Multi Tissue Dissociation Kit 2.The cell yield was quantified using a high-content imaging analysis system by counting the number of adherent cells per field after 72 h of culture.The CFs purity was assessed using immuno- fluorescence staining for vimentin.The trans-differentiation activity of the CFs was evaluated with trans- forming growth factor β1（TGF-β1）. Results：Omitting any component of the digestion solution（colla- genase II/IV ，trypsin or DNase I），significantly prolonged extraction time and reduced cell yield.In contrast，the optimized protocol outperformed the commercial kit，reducing digestion time by 32.2 min and significantly increasing cell yield，and with comparable obtained CFs purity.After TGF-β1 stimula- tion，CFs exhibited enhanced proliferative capacity and upregulated expression of α-smooth muscle actin （α-SMA），collagen type I（Col I），and fibronectin（FN），confirming the differentiation potential of CFs isolated via the optimized method. Conclusion：This study systematically optimized an enzymatic di-gestion method combining collagenase，trypsin，and nuclease in conjunction with mechanical dissociation using a tissue dissociator，leading to the efficient and stable isolation of adult mouse CFs.By fine-tuning enzyme concentrations and digestion conditions，we successfully reduced processing time，improved cell yield，and enhanced cell viability compared with conventional isolation methods.These findings validate the physiological relevance of the isolated CFs and demonstrate that the optimized protocol provides a reliable and reproducible method for studying myocardial fibrosis and remodeling.This protocol can serve as a valuable tool for researchers investigating CFs biology and its role in cardiovascular diseases.

关键词

小鼠 / 心脏成纤维细胞 / 原代细胞培养 / 组织解离

Key words

Mice / Cardiac fibroblasts / Primary cell culture / Tissue dissociation

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基金资助

国家自然科学基金(82471329)