Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy

Abstract

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.