Advancing practical usage of microtechnology: a study of the functional consequences of dielectrophoresis on neural stem cells

Abstract

The integration of microscale engineering, microfluidics, and AC electrokinetics such as dielectrophoresis has generated novel microsystems that enable quantitative analysis of cellular phenotype, function, and physiology. These systems are increasingly being used to assess diverse cell types, such as stem cells, so it becomes critical to thoroughly evaluate whether the systems themselves impact cell function. For example, engineered microsystems have been utilized to investigate neural stem/progenitor cells (NSPCs), which are of interest due to their potential to treat CNS disease and injury. Analysis by dielectrophoresis (DEP) microsystems determined that unlabeled NSPCs with distinct fate potential have previously unrecognized distinguishing electrophysiological characteristics, suggesting that NSPCs could be isolated by DEP microsystems without the use of cell type specific labels. To gauge the potential impact of DEP sorting on NSPCs, we investigated whether electric field exposure of varying times affected survival, proliferation, or fate potential of NSPCs in suspension. We found short-term DEP exposure (1 min or less) had no effect on NSPC survival, proliferation, or fate potential revealed by differentiation. Moreover, NSPC proliferation (measured by DNA synthesis and cell cycle kinetics) and fate potential were not altered by any length of DEP exposure (up to 30 min). However, lengthy exposure (> 5 min) to frequencies near the crossover frequency (50–100 kHz) led to decreased survival of NSPCs (maximum ~30% cell loss after 30 min). Based on experimental observations and mathematical simulations of cells in suspension, we find that frequencies near the crossover frequency generate an induced transmembrane potential that results in cell swelling and rupture. This is in contrast to the case for adherent cells since negative DEP frequencies lower than the crossover frequency generate the highest induced transmembrane potential and damage for these cells. We clarify contrasting effects of DEP on adherent and suspended cells, which are related to the cell position within the electric field and the strength of the electric field at specific distances from the electrodes. Modeling of electrode configurations predicts optimal designs to induce cell movement by DEP while limiting the induced transmembrane potential. We find DEP electric fields are not harmful to stem cells in suspension at short exposure times, thus providing a basis for developing DEP-based applications for stem cells.

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

“…13,18 In some cases, NSPCs were isolated from E16 cortices to provide cells from a different developmental stage. Prior to sorting, NSPC neurospheres were dissociated using NeuroCult Chemical Dissociation kit (Stem Cell Technologies) and resuspended in DEP buffer, which is low conductivity (110 lS/cm) to enable sorting using positive DEP.…”

“…Cells were placed in DEP wells and electrodes were actuated at either 100 kHz (sorted) or 1000 kHz (control) with a 3 V pp signal for 5 min or less to prevent toxicity. 13 Cells not in positive DEP were removed with three washes of DEP buffer. After the washes, the function generator was turned off to release the cells, which were then collected from the well.…”

“…13 E12 or E16 mNSPC neurospheres were dissociated using NeuroCult and resuspended in DEP buffer as described above to a final concentration of $3 Â 10 6 cells/ml. Cells were placed in DEP wells and electrodes were actuated at either 100 kHz (sorted) or 1000 kHz (control) with a 3 V pp signal for 5 min or less to prevent toxicity.…”

“…10,13 Briefly, 25 mm  75 mm glass slides were coated with 200 Å titanium, followed by 1000 Å gold, and the electrode features were patterned with AZ 4620 photoresist (AZ Electronic Materials, Branchburg, NJ, USA). Following patterning, the gold and titanium were selectively etched, and the sacrificial AZ photoresist layer was stripped to yield the electrode features on the glass slide.…”

“…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. 13 The fact that several types of stem and progenitor cells have been sorted by DEP without deleterious effects on the cells shows the promise of this technique for stem cell isolation.…”