Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle

Chibeza C. Agley*, Anthea M. Rowlerson, Cristiana P. Velloso, Norman L. Lazarus, Stephen D R Harridge

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

34 Citations (Scopus)

Abstract

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+and later as CD56+/desmin+cells and (ii) muscle-derived fibroblasts, identified as CD56and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 106± 8.87 x 105cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+cells bound to microbeads are retained by the field whereas CD56cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

Original languageEnglish
Article numbere52049
JournalJournal of Visualized Experiments
Issue number95
DOIs
Publication statusPublished - 12 Jan 2015

Keywords

  • Adipocytes
  • Developmental biology
  • Fibroblasts
  • Image analysis
  • Issue 95
  • Magnetic activated cell sorting
  • Myoblasts
  • Myogenic cells
  • Satellite cells
  • Skeletal muscle
  • Stem cell biology
  • Stem cells
  • Tissue Engineering

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