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

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.

Frequency discretization in dielectrophoretic assisted cell sorting arrays to isolate neural cells.

We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.

Biophysical characteristics reveal neural stem cell differentiation potential.

BACKGROUND: Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. CONCLUSIONS/SIGNIFICANCE: We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq.

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%-50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3' untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.

Engineering microscale cellular niches for three-dimensional multicellular co-cultures.

Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell-cell and cell-matrix interactions.

Dual frequency dielectrophoresis with interdigitated sidewall electrodes for microfluidic flow-through separation of beads and cells.

This paper presents a novel design and separation strategy for lateral flow-through separation of cells/particles in microfluidics by dual frequency coupled dielectrophoresis (DEP) forces enabled by vertical interdigitated electrodes embedded in the channel sidewalls. Unlike field-flow-fractionation-DEP separations in microfluidics, which utilize planar electrodes on the microchannel floor to generate a DEP force to balance the gravitational force and separate objects at different height locations, lateral separation is enabled by sidewall interdigitated electrodes that are used to generate non-uniform electric fields and balanced DEP forces along the width of the microchannel. In the current design, two separate AC electric fields are applied to two sets of independent interdigitated electrode arrays fabricated in the sidewalls of the microchannel to generate differential DEP forces that act on the cells/particles flowing through. Individual particles (cells or beads) will experience DEP forces differently due to the difference in their dielectric properties. The balance of the differential DEP forces from the electrode arrays will position dissimilar particles at distinct equilibrium planes across the width of the channel. When coupled with fluid flow, this results in lateral separation along the width of the microchannel and the separated particles can thus be automatically directed into branched channel outlets leading to different reservoirs for downstream processing. In this paper, we present the design and analysis of lateral separation enabled by dual frequency coupled DEP, and cell/bead and cell/cell separations are demonstrated with this lateral separation strategy. With vertical interdigitated electrodes on the sidewall, the height of the microchannel can be increased without losing the electric field strength in contrast to other multiple frequency DEP devices with planar electrodes. As a result, populations of cells can be separated simultaneously instead of one by one to enable high-throughput sorting microfluidic devices.

Unique dielectric properties distinguish stem cells and their differentiated progeny.

The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers, such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting, are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP), which induces a frequency-dependent dipole in cells, would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs), differentiated neurons, and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons, or astrocytes, we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition, experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating, characterizing, and sorting stem cells.

A novel oscillating bioreactor BelloCell: implications for insect cell culture and recombinant protein production.

BelloCell is a novel packed bed bioreactor that allows alternating nutrient and gas transfer to a culture. Spodoptera frugiperda Sf-9 grown in the BelloCell (300 ml culture) reached 1.3-1.5x10(7) cells ml-1 in 7-8 days and the total baculovirus-expressed protein yield was 2.3-times that in a stirred tank bioreactor (600 ml culture). The superior cell and protein yields underline the potential of BelloCell for cell culture and recombinant protein production.

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor.

A novel two-compartment bioreactor, BelloCell((R)), was used to cultivate insect cells and a maximum yield of 4.6 x 10(9) cells was attained. The cells were immobilized in a packed bed fixed in the upper chamber, and the bellow in the lower chamber was compressed and released in an alternating fashion. The motion resulted in gentle, cyclic movement of the medium that was contained in the lower chamber and consequently exposed the cells to air in an oscillatory manner, thus rendering adequate aeration and uniform cell distribution in the bed. The baculovirus yield produced in BelloCell((R)) could amount up to 3.3 x 10(17) pfu using as little as 1.1 l medium in the production run. Besides, BelloCell((R)) was extremely easy to handle and operate. These benefits underline the potential of BelloCell((R)) for simple, economical and high-density cell culture and protein/virus production.