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 lm). 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.

Hydrodynamic mechanisms of cell and particle trapping in microfluidics

“…18 The controlling mechanism in the aforementioned handling techniques can be classified in two categories: active methods based on the application of external force fields and passive methods where their functionality is established by harnessing microchannel geometrical effects and nonlinear hydrodynamic forces. In the past decade, extensive investigations have been conducted in order to trap and sort cells and particles using either innovative active techniques, such as dielectrophoresis (DEP), [19][20][21][22] magnetophoresis, 23 acoustophoresis, 24 and optical tweezers, 25 or novel passive approaches, including pinched flow fractionation (PFF), 26 hydrodynamic filtration, 27 biomimetic methods, 28 hydrophoretic focusing, 29 deterministic lateral displacement (DLD), 30 and surface acoustic wave (SAW)-induced streaming. 31 Each of these methods is associated with some advantages and deficiencies which make them preferable in certain applications.…”

Increasing label-free stem cell sorting capacity to reach transplantation-scale throughput

“…7 Several types of stem and progenitor cells (hematopoietic, adipose-derived progenitors, and myoblasts) have been successfully analyzed by flow cytometry after DEP sorting. [7][8][9]11 Furthermore, DEP sorted myoblasts have high viability post-sorting. 11 Mouse NSPCs isolated by DEP were analyzed in differentiation assays, demonstrating that the cells retained the ability to generate neurons and astrocytes post-DEP sorting.…”

“…[7][8][9]11 Furthermore, DEP sorted myoblasts have high viability post-sorting. 11 Mouse NSPCs isolated by DEP were analyzed in differentiation assays, demonstrating that the cells retained the ability to generate neurons and astrocytes post-DEP sorting. 12 Taken together, the fact that several studies demonstrate viable stem cells after DEP sorting suggest that other stem cell populations could be expanded post sorting in order to generate sufficient numbers of cells for experiments such as transplantation.…”

“…9 DEP-based separation can isolate undifferentiated from more differentiated cells in the same lineage, as shown by the separation of neural stem and progenitor cells (NSPCs) from differentiated neurons 10 and separation of C2C12 myoblasts and more differentiated myotubes. 11 Progenitor cells within the same lineage are also amenable to separation using DEP, and enrichment of neuron progenitors and astrocyte progenitors from a mixed population of NSPCs by DEP provides significantly better enrichment than FACS sorting with PSA-NCAM-a purported marker for neuron progenitors. 12 Sorting by DEP is not toxic for NSPCs, since exposure of these cells to DEP electric fields for the times needed for sorting does not alter cell survival, proliferation, or differentiation.…”

Efficient dielectrophoretic cell enrichment using a dielectrophoresis-well based system

“…6 Since then, many researchers have developed systems to investigate the separation of circulating tumour cells from blood samples (e.g., Ref. 7); discriminate between, and subsequently separate, stem cell populations with different differentiation potential; [8][9][10] remove diesel particulate matter from airborne samples whilst retaining airborne bacteria; 11 as well as multiple separations of live and dead cells such as yeast.…”