Optimizing Neural Stem Cell Sorting with Dielectrophoresis

Human neural stem and progenitor cells (NSPCs) have therapeutic potential to treat neurodegenerative diseases since they provide neuroprotection and differentiate into the three cell types of the central nervous system - astrocytes, neurons, and oligodendrocytes. However, cultures of these cells are heterogeneous, containing progenitor cells with distinct differentiation properties and little is known regarding which types of progenitors are best for neural repair. Dielectrophoresis (DEP) is a technique that uses alternating current (AC) electric fields to separate cells based on the dielectric properties of their membrane and cytoplasm and has recently shown promise for separating progenitors in the neural lineage.

            Using DEP, our group has demonstrated that astrocyte progenitor (AP) and neuron progenitor (NP) cells can be isolated from mouse NSPCs at select frequencies and this behavior is linked to the cells’ membrane capacitance and fate potential.  We also discovered that NSPCs DEP response was not always the same at a single frequency, which can be attributed to variations in cell populations, microdevices, or media conductivity. To remedy these variations, we’ve implemented a two-step sorting scheme for NSPCs, which can be extended to other cell systems. Step one defines a cell sample specific trapping frequency, and step two separates cells at that specific frequency in a DEP well microdevice.

            In this work human AP cells were detected and separated from heterogeneous NSPCs using DEP. A trapping frequency specific to AP cells ranging from 8-20kHz was defined using a 3DEP reader. Then, a multi-well DEP device with Ti-Au interdigitated electrodes was used to sort AP cells at the trapping frequency. Prior to sorting, a mini-trapping curve was developed over the frequency range 5-100kHz in the multi-well DEP device, confirming acceptable accuracy of the predicted trapping frequency. For post-sorting analysis, cell differentiation and 3DEP reader analysis were completed to verify AP separation, determine cell enrichment, and measure membrane capacitance values of sorted cells.  Cells trapped at 1MHz and incubated in the DEP buffer solution were tested as controls. AP separation was confirmed with GFAP immunostaining to detect astrocytes differentiated from APs and the cell enrichment was found to be 1.75 fold. Effectively sorting human NSPCs is essential to further study these cells as treatment options for neurodegenerative diseases.