Primary culture and characterization of microvascular endothelial cells from murine skeletal muscle with immuomagnetic beads

  AIM   To establish an accurate and effective technique and method of isolating and culturing mouse skeletal muscle microvascular endothelial cells (MMECs) in vitro.

  METHODS   Mouse MMECs were isolated and purified by collagenase I digesting and differential adhesion combined with magnetic beads and were adherently cultured in vitro. The cell morphology and ultrastructure were observed by microscopes, the growth curve of the cultured MMECs was measured by MTT, its phenotype was identified by CD31 related antigen immunofluorescence staining, and the purity of the MMECs was detected by flow cytometry.

  RESULTS   Mouse skeletal muscle microvascular endothelial cells were obtained successfully and the primary cultured endothelial cells quickly adhered to the wall after 24 hours of culture, grew actively in monolayer with the characteristics of endothelial cell shape at (3~6) days of culture and fully covered the bottom in a “cobblestone” arrangement after (6~8) days of culture. MTT assay showed that cell growth curves of 2 generations of MMECs presented the inverted “S” shape. The results of flow cytometry indicated that the positive rate of CD31⁺ labelled MMECs purified with differential adhesion plus magnetic beads was over 97%.

  CONCLUSION   A simple, reproducible and well tested method is established for isolating microvascular cells of murine skeletal muscle, which can provide the material basis and experimental model for further research on microvascular-related myopathy and molecular biology in related fields.