Isolation，culture，and identification of mesenchymal stem cells derived from rat abdominal aorta based on the enzymatic digestion-tissue explant sequential method

Lai Suyu; Wang Yichang; Liu Chenxuan; Cai Chaoying; Jiang Yun; He Yinghua; Wang Xiaofeng

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Isolation，culture，and identification of mesenchymal stem cells derived from rat abdominal aorta based on the enzymatic digestion-tissue explant sequential method

更新时间：2026-06-10

- Isolation，culture，and identification of mesenchymal stem cells derived from rat abdominal aorta based on the enzymatic digestion-tissue explant sequential method
- Journal of Chongqing Medical University Pages: 1-7(2026)
- 作者机构：
  
  中国中医科学院广安门医院肛肠科，北京 100053
- 作者简介：
- 基金信息：
- DOI：
  CLC： R-33
- Received：20 November 2025，
  
  Revised：2026-01-21，
  
  Accepted：25 February 2026，
  
  Online First：10 June 2026，
- 稿件说明：
移动端阅览
Lai Suyu,Wang Yichang,Liu Chenxuan,et al.Isolation，culture，and identification of mesenchymal stem cells derived from rat abdominal aorta based on the enzymatic digestion-tissue explant sequential method[J].Journal of Chongqing Medical University,
DOI：

Lai Suyu,Wang Yichang,Liu Chenxuan,et al.Isolation，culture，and identification of mesenchymal stem cells derived from rat abdominal aorta based on the enzymatic digestion-tissue explant sequential method[J].Journal of Chongqing Medical University, DOI：10.13406/j.cnki.cyxb..

摘要

目的

建立优化的“酶解-组织块序贯法”，以实现大鼠腹主动脉来源间充质干细胞（mesenchymal stem cells，MSCs）的高效分离、扩增与系统鉴定。

方法

采用3月龄SD大鼠，无菌分离腹主动脉并剪碎，依次经0.1% Ⅰ型胶原酶有限消化及组织块迁移培养法进行原代细胞获取。采用倒置显微镜动态观察细胞形态与生长特性，采用流式细胞术检测第4代细胞表面标志物［分化抗原簇（cluster of differentiation，CD）73、CD44、CD29、CD34、CD45、CD11b/c］的表达，并通过成脂、成骨及成软骨三系诱导分化实验评估其多向分化潜能。

结果

原代细胞在第3天即开始从组织块周围迁出，增殖旺盛，传代后呈典型的“漩涡样”生长。细胞高表达CD73（95.9±1.5）%、CD44（94.5±0.8）%与CD29（82.5±1.8）%；同时，低表达造血系统标志物CD45（13.0±3.2）%、CD34（1.65±0.5）%及CD11b/c（8.6±2.1）%。经定向诱导后，细胞可形成油红O染色阳性的脂滴、茜素红染色阳性的钙结节以及阿利新蓝染色阳性的蛋白聚糖，证实其具备完整的三系分化能力。与传统单纯组织块法或单纯酶解法相比，本序贯法在细胞迁出时间、原代融合效率方面具有优势。

结论

“酶解-组织块序贯法”是一种高效、稳定的腹主动脉MSCs分离培养体系，所获细胞纯度高、干性维持良好、多向分化潜能完备，为血管相关疾病的研究与细胞治疗提供了优质的种子细胞来源。

Abstract

Objective

To establish an optimized enzymatic digestion-tissue explant sequential method，and to realize efficient isolation，expansion，and systematic identification of mesenchymal stem cells（MSCs） derived from rat abdominal aorta.

Methods

Sprague-Dawley rats，aged 3 months，were selected，and abdominal aortas were aseptically isolated and minced，followed by primary cell harvesting via sequential limited digestion with 0.1% type Ⅰcollagenase and explant migration culture. An inverted microscope was used for dynamic observation of cell morphology and growth；flow cytometry was used to measure the expression of surface markers （CD73，CD44，CD29，CD34，CD45，and CD11b/c） on passage 4 cells；adipogenic，osteogenic，and chondrogenic trilineage differentiation experiments were used to assess multidirectional differentiation potential.

Results

Primary cells began migrating from the tissue explants on day 3，exhibiting vigorous proliferation and forming a typical “whirlpool-like” growth pattern after passaging. The cells showed high expression levels of CD73［（95.9±1.5）%］，CD44［（94.5±0.8）%］，and CD29［（82.5±1.8）%］ and low expression levels of the hematopoietic markers CD45［（13.0±3.2）%］，CD34［（1.65±0.5）%］，and CD11b/c［（8.6±2.1）%］. Upon directed induction，the cells formed lipid droplets positive for oil red O staining，calcium nodules positive for alizarin red staining，and proteoglycans positive for Alcian blue staining，which confirmed the complete trilineage differentiation capacity of MSCs. Compared with the traditional tissue explant method or the enzymatic digestion method alone，this sequential method showed advantages in initial cell migration time and primary confluence efficiency.

Conclusion

The enzymatic digestion-tissue explant sequential method is an efficient and stable system for isolating and culturing MSCs derived from the abdominal aorta，and the cells obtained have high purity，well-maintained stemness，and complete multilineage differentiation potential，which provides a high-quality seed cell source for research on vascular diseases and cell therapy.

关键词

Keywords

references

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