Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2022)

Cell heterogeneity is essential in organism development and is a key feature in many diseases. Identifying the defining features of distinct cell types within a heterogeneous population can improve our understanding of development and disease progression and lead to the discovery of better therapeutics. Conventional label-based cell separation methods are often used to isolate distinct cell types from a heterogeneous population. However, this approach has a number of disadvantages, including a lack of unique biomarkers to identify cells of interest. This hampers mechanistic studies aimed at deciphering the unique properties of specific cell types in heterogeneous cell populations. Label-free cell separation techniques generate enriched cell populations necessary for investigating distinct cell functions without the need for unique cell type-specific labels. This body of work focuses on the utilization of dielectrophoresis (DEP), a label-free electrokinetic method, to study heterogeneous cell populations. It details the development of the Hydrophoretic Oblique Angle Parallel Electrode Sorting (HOAPES) device, a high-throughput DEP-based cell sorter that can separate cells based on intrinsic electrical properties. This novel cell sorter combined multiple microfluidic modules to enable continuous separation of distinct cell types and was used to analyze heterogeneous populations of neural stem and progenitor cells (NSPCs) and glioblastoma (GBM) cells. The HOAPES device utilizes continuous fluid flow to enable high throughput separation, providing sufficient cells for downstream assays to assess both sorting performance and identify distinct cell properties associated with sorted cell phenotype. Furthermore, this method helped uncover correlations between cell fate, cell surface N-glycosylation and electrophysiological properties of NSPCs. Similarly, links were identified between chemotherapeutic resistance, glycosylation, and membrane electrophysiological properties of GBM cells.

    Creative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2018)
    Reliable separation and isolation of target single cells from bodily fluids with high purity is of great significance for an accurate and quantitative understanding of the cellular heterogeneity. Here, we describe a fully integrated single-blood-cell analysis platform capable of size-selective cell separation from a population containing a wide distribution of sizes such as diluted blood sample and highly efficient entrapment of single monocytes. The spiked single U937 cells (human monocyte cell line) are separated in sequence by two different-sized microfilters for removing large cell clumps, white blood cells, and red blood cells and then discriminated by dielectrophoretic force and isolated individually by downstream single-cell trapping arrays. When 2% hematocrit blood cells with a final ratio of 1:1000 U937 cells were introduced under the flow rate of 0.2 ml/h, 400 U937 cells were trapped sequentially and deterministically within 40 s with single-cell occupancy of up to 85%. As a proof-of-concept, we also demonstrated single monocyte isolation from diluted blood using the integrated microfluidic device. This size-selective, label-free, and live-cell enrichment microfluidic single blood-cell isolation platform for the processing of cancer and blood cells has a myriad of applications in areas such as single-cell genetic analysis, stem cell biology, point-of-care diagnostics, and cancer diagnostics.
      Cover page: An integrated microfluidic platform for size-selective single-cell trapping of monocytes from bloodCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2018)
      Whole cell membrane capacitance is an electrophysiological property of the plasma membrane that serves as a biomarker for stem cell fate potential. Neural stem and progenitor cells (NSPCs) that differ in ability to form neurons or astrocytes are distinguished by membrane capacitance measured by dielectrophoresis (DEP). Differences in membrane capacitance are sufficient to enable the enrichment of neuron- or astrocyte-forming cells by DEP, showing the separation of stem cells on the basis of fate potential by membrane capacitance. NSPCs sorted by DEP need not be labeled and do not experience toxic effects from the sorting procedure. Other stem cell populations also display shifts in membrane capacitance as cells differentiate to a particular fate, clarifying the value of sorting a variety of stem cell types by capacitance. Here, we describe methods developed by our lab for separating NSPCs on the basis of capacitance using several types of DEP microfluidic devices, providing basic information on the sorting procedure as well as specific advantages and disadvantages of each device.
        Cover page: Separation of neural stem cells by whole cell membrane capacitance using dielectrophoresis
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2019)
        We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort.
          Cover page: High-throughput continuous dielectrophoretic separation of neural stem cells.Creative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2019)
          Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.
            Cover page: Identification of neural stem and progenitor cell subpopulations using DC insulator-based dielectrophoresis.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2020)
            Human neural stem and progenitor cells (hNSPCs) have therapeutic potential to treat neural diseases and injuries since they provide neuroprotection and differentiate into astrocytes, neurons, and oligodendrocytes. However, cultures of hNSPCs are heterogeneous, containing cells linked to distinct differentiated cell fates. HNSPCs that differentiate into astrocytes are of interest for specific neurological diseases, creating a need for approaches that can detect and isolate these cells. Astrocyte-biased hNSPCs differ from other cell types in electrophysiological properties, namely membrane capacitance, and we hypothesized that this could be used to enrich these cells using dielectrophoresis (DEP). We implemented a two-step DEP sorting scheme, consisting of analysis to define the optimal sorting frequency followed by separation of cells at that frequency, to test whether astrocyte-biased cells could be separated from the other cell types present in hNSPC cultures. We developed a novel device that increased sorting reproducibility and provided both enriched and depleted cell populations in a single sort. Astrocyte-biased cells were successfully enriched from hNSPC cultures by DEP sorting, making this the first study to use electrophysiological properties for label-free enrichment of human astrocyte-biased cells. Enriched astrocyte-biased human cells enable future experiments to determine the specific properties of these important cells and test their therapeutic efficacy in animal models of neurological diseases.
              Cover page: Label-free enrichment of fate-biased human neural stem and progenitor cells
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2019)
              Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.
                Cover page: Identification of neural stem and progenitor cell subpopulations using DC insulator-based dielectrophoresis
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2016)
                We present a microfluidic lab-on-printed circuit board (PCB) device containing alloy vertical electrodes for sorting microparticles using dielectrophoresis. The device consists of a hydrodynamic prefocuser and an electronic sorting region. Lining the two sidewalls of the electronic sorting region are regularly spaced rectangular metal electrodes reaching from the floor to the ceiling of the flow channel that bridge electric field lines laterally across the channel. The size and distribution of these vertical electrodes are arranged asymmetrically such that the resultant electric field forms sharp electric field gradients across the channel; specific geometries were optimized using finite element methods. Particles entering the device are initially focused on a single stream as they pass through the prefocuser. Subsequently, they are exposed to the lateral electric field gradient and separate into streams based on their size and dielectric properties. Validation was performed by dielectrophoretically separating live cells from dead cells. Importantly, the system presented can be readily integrated with various external sensors and actuators using commercially available components owing to the device's integration into a PCB.
                  Cover page: 3-D In-Bi-Sn Electrodes for Lab-on-PCB Cell Sorting
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2018)
                  Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.
                    Cover page: Cell Surface N-Glycans Influence Electrophysiological Properties and Fate Potential of Neural Stem Cells.Creative Commons 'BY' version 4.0 license
                    Top