[go: up one dir, main page]

WO2015017729A1 - Procédés de diélectrophorèse pour déterminer une propriété d'une pluralité de cellules cancéreuses - Google Patents

Procédés de diélectrophorèse pour déterminer une propriété d'une pluralité de cellules cancéreuses Download PDF

Info

Publication number
WO2015017729A1
WO2015017729A1 PCT/US2014/049296 US2014049296W WO2015017729A1 WO 2015017729 A1 WO2015017729 A1 WO 2015017729A1 US 2014049296 W US2014049296 W US 2014049296W WO 2015017729 A1 WO2015017729 A1 WO 2015017729A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hydrochloride
cancer
drug
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/049296
Other languages
English (en)
Inventor
Eva M. Schmelz
Elizabeth S. ELVINGTON
Alireza SALMANZADEH
Mike SANO
Paul C. Roberts
Rafael V. Davalos
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Virginia Tech Intellectual Properties Inc
Original Assignee
Virginia Tech Intellectual Properties Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Virginia Tech Intellectual Properties Inc filed Critical Virginia Tech Intellectual Properties Inc
Publication of WO2015017729A1 publication Critical patent/WO2015017729A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications

Definitions

  • the present disclosure relates to devices and methods for dielectrophoresis (DEP) for manipulation of cells or particles. More particularly, the present disclosure relates to methods for determining one or more intrinsic electrical properties of a plurality of cancer cells through dielectrophoresis, such as by using contactless dielectrophoresis.
  • DEP dielectrophoresis
  • Ovarian cancer is the most common cause of death from gynecological malignancies and one of the top causes of cancer-related deaths of women in the United States and Europe [1 , 2].
  • the relative 5-year survival rate for invasive epithelial ovarian cancer patients diagnosed at early stages is more than 90%, but drops to less than 30% when the disease is first diagnosed at later stages [3A], highlighting the importance of early detection and the need for improved treatment options.
  • Chemotherapeutic regimens typically conform to institutional protocols and subjective experiences of individual physicians, however, responses to these protocols can vary greatly between patients. This is largely due to the difficulty in assessing an individual's response to a particular regimen prior to treatment. More than 65% of women diagnosed with Stage III or later ovarian cancer will experience recurrence of the disease and will need to undergo additional rounds of chemotherapy [4A]. Chemotherapeutic resistance, a common occurrence in progressive disease [5A], can limit the success of consecutive rounds of therapy in the treatment of any type of cancer. There are a number of pharmacological, physiological, and molecular mechanisms associated with this resistance, however, the exact mechanisms which cause resistance are still unresolved.
  • Ovarian cancer is a heterogeneous disease with a broad genetic and epigenetic profile, various histological manifestations and even different origins.
  • Current conventional chemotherapeutic drugs targeting oncogenes such as Her-2 or VEGFR are successful in less than 30% of patients with aberrant expression of these targets, require chemotherapeutic dosages that are highly toxic to non-transformed cells, and/or induce drug resistance in a sub-population of cancer cells causing disease recurrence within 5-6 months.
  • This heterogeneity would require the determination of the individual geno/phenotype for more successful treatment decisions that can enhance the survival of afflicted women.
  • the bioelectrical properties of cancer cells will allow for detection of early stages of ovarian cancer independent of specific molecular markers.
  • the bioelectrical signature may be unique to the cancer cell and/or its stem-like population, and the response to treatment. It is thus expected that the drug resistant phenotype of cells will impart a unique bioelectrical signature (otherwise referred to as a bioelectrical fingerprint in this disclosure) that will enable early identification of drug resistant cells that may also be exploited to monitor the development of drug resistance during tumor recurrence.
  • the bioelectrical signature of cells can be determined by dielectrophoresis (DEP), the motion of a polarizable particle in a non-uniform electric field [9A, 10A].
  • DEP is successful technique for nondestructive manipulation [1 1 ], separation [12A], and detection [13A] of bioparticles in microdevices, based on their size, shape, internal structure, and electrical properties such as conductivity and polarizability.
  • DEP does not require the use of target-specific antibodies and has been shown to successfully isolate cancer cells from blood [16A, 17A, 18A], and from CD34+ hematopoietic stem cells [19A].
  • DEP could overcome the limitation of current approaches and provide a platform to assess individual drug responsiveness and metastatic potential. The inventors envision that this approach can be also applied to other cancer types and uses such as cancer cell detection.
  • Embodiments of the present disclosure provide a method of determining a property of a plurality of cancer cells.
  • the method comprises providing a dielectrophoresis device comprising a sample channel for receiving a sample.
  • the device comprises a sample channel or series of separating channels with a separating portion; a first electrode channel for receiving a first electrode; a first insulation barrier between the first electrode channel and the sample channel; a second electrode channel for receiving a second electrode; and a second insulation barrier between the second electrode channel and the sample channel.
  • Other embodiments may not require a separate channel to contain the fluid portion.
  • the method comprises providing a plurality of cancer cells, such as in a cancer cell suspension, introducing the cancer cell suspension to the sample channel, generating an AC electric field through the first and second electrode, and measuring the spatial distribution of cells through the sample channel, wherein the spatial distribution is characteristic of a property of the cancer cells.
  • the spatial distribution response can be used to identify and differentiate cancer cells from one another and/or characterize them, or interrogate, separate, or enrich them for further selective treatment of certain cells, such as downstream characterization protocols as needed, including for example, Genomics, Biochemical profiles, etc.
  • FIG. 1 is graph showing crossover frequency for MOSE cells.
  • the FFL stem cell electrical signature increases compared to the MOSE-L parent stage cells.
  • FIG. 2 is a schematic diagram showing a cDEP Device Schematic.
  • FIGS. 3A and 3B are images showing MOSE cells experiencing negative and positive DEP, respectively.
  • FIG. 4 is a diagram showing a summary of cancer cell separation studies.
  • FIG. 5 is a schematic diagram showing an overhead view schematic of an exemplary low frequency single-layer microfluidic device used for characterizing MOSE cells.
  • the inset detail view shows a sawtooth feature and the thin insulating barrier separating sample channel and electrode channels.
  • FIG. 6A is a schematic diagram showing a surface plot for computational modeling of * x ⁇ M ⁇ ' ⁇ MSJ j n the sample channel of a multilayer cDEP device with overlaid curved tapered electrode channels.
  • the plot was generated at 100 V, 200 kHz.
  • the legend (units of Hz) shows the frequency range 100-500 kHz.
  • FIG. 7 A is a schematic diagram showing a surface plot for computational modeling of * Jt S * - j n t e sample channel of a multilayer cDEP device with curved tapered electrode channels.
  • FIG. 7B is a graph showing a line plot along the x-centerline of the sample channel demonstrating the change in * l to s i!f Iii; with frequency.
  • the legend shows the frequency value in Hz.
  • FIGS. 8A-C are illustrations showing computational modeling of samples channel: (a) shear rate, (b) electric field magnitude, and (c) * l ⁇ i' ⁇ liW,
  • FIGS. 9A and 9B are schematic diagrams of predictions of the particles trajectories at (a) 5 kHz and (b) 20 kHz in red lines for 10 particles. Trajectories appear to diminish down the channel due to a simulation artifact that occurs when trajectories encounter a wall. i ⁇ ⁇ " " $ s also presented in the background. Darker areas indicate higher * * ⁇ * 3 ⁇ 4 ⁇ The scale bar represents 500 ⁇ .
  • FIGS. 10A-C are images showing MOSE-L cell movement in the sample channel (a) in control conditions under no external electric field, due to applying 200 VRMS and (b) negative DEP force, 5 kHz and (c) positive DEP force, 30kHz.
  • FIGS. 10D-F are plots showing normalized cells distribution corresponding to (d) control distribution in (a), (e) negative DEP from (b), and (f) positive DEP from (c).
  • FIGS 1 1 A and 1 1 B are graphs showing that SL-treated late stage cells revert back to early stage based on their electrical signature, (a) m and
  • FIGS. 12A-12C are graphs showing computational modeling of
  • ⁇ increases with increasing y-position in the channel due to narrowing width of the electrode channels.
  • the present disclosure provides methods, devices, and systems to manipulate micro-particles suspended in biological fluids (including solid tumors in suspension) using their electrical characteristics signatures.
  • such methods can be performed in the absence of direct contact between the electrodes and the sample.
  • Contactless dielectrophoresis employs the simplified fabrication processes of iDEP yet lacks the problems associated with the electrode- sample contact as in DEP [40A].
  • Methods of the invention can also be used with DEP and/or iDEP.
  • DEP uses a solid metal electrode that does not require a separate channel as cDEP does to contain the fluid portion.
  • cDEP relies upon reservoirs filled with highly conductive fluid to act as electrodes and provide the necessary electric field. These reservoirs are placed adjacent to the main microfluidic channel and are separated from the sample by a thin barrier of a dielectric material. The application of a high-frequency electric field to the electrode reservoirs causes their capacitive coupling to the main channel and an electric field is induced across the sample fluid.
  • cDEP may exploit the varying geometry of the electrodes to create spatial non-uniformities in the electric field.
  • the electrode structures employed by cDEP can be fabricated in the same step as the rest of the device; hence the process is conducive to mass production [40A].
  • Various embodiments of the present disclosure provide devices and methods for performing cDEP, iDEP, and/or DEP, as well as methods for fabricating such devices.
  • the present disclosure provides devices and methods that allow cell sorting to identify, isolate or otherwise enrich cells of interest based on electrical and physical properties of the target cells.
  • an electric field is induced in a sample comprising the target cells.
  • the cells are cancer cells, but any cell may be used.
  • any particle of size which would fit down the channel such as DNA, RNA, cell particles, protein, viruses, and bio-markers can also be used.
  • the electric field can be induced in a main sorting microchannel using electrodes inserted in a highly conductive solution which is isolated from the microchannel by thin insulating barriers. The insulating barriers exhibit a capacitive behavior and an electric field is produced in the isolated microchannel by applying an AC electric field.
  • the device Although it is possible to design the device so that electrodes contact the sample fluid, a contactless approach is preferred in which the electrodes do not come into contact with the sample fluid inside the microchannel. With no contact between the electrodes and sample fluid, electrolysis, bubble formation, fouling and contamination can be reduced or eliminated. In addition, the electric field is focused in a confined region and has a much lower intensity than that found in traditional insulator-based dielectrophoresis, so heating within the sample channel is negligible and the likelihood of cell lysis is greatly reduced.
  • the system can also be used for characterizing and sorting micro- or nanoparticles.
  • the present disclosure provides devices and methods that allow for the determination of a cancer cells sensitivity or resistance to a cancer therapeutic agent (e.g., chemotherapeutic drugs), or, alternatively, the determination of malignant properties of the cancer cells such as the stage of transformation, tumorigenicity and metastatic potential, and stem-like properties, based on the electrical and physical properties of the cancer cell.
  • a cancer therapeutic agent e.g., chemotherapeutic drugs
  • the present disclosure provides devices and methods that allow for the detection and enrichment of drug-resistant cells by their altered bioelectrical fingerprint independent of their specific genotype and origin.
  • the devices and methods can be used to define and characterize "specific" drug responsiveness and drug resistance that can be used to prescreen tumor cells of a particular patient for resistance (for example, drug resistant populations display an altered bioelectrical profile) prior to beginning a treatment regimen on that patient, including conventional drugs and novel experimental drugs and treatments.
  • the devices and methods can be used for monitoring of treatment for efficacy and direct further treatment decisions/modifications of treatment.
  • the bioelectrical fingerprint is phenotype dependent.
  • the present disclosure provides devices and methods that allow for characterization of cells by their bioelectrical fingerprint, including by their "dielectrophoretic response".
  • dielectrophoretic response means the intrinsic electrical characteristics of a cell; dependent upon its size, shape morphology and topography, including the internal organelles (nucleus size, polidity, endoplasmic reticulum) and structures, thickness of the lipid bilayer or cell membrane, nucleus size endoplasmic reticulum, and ratio of internal structures to cytoplasm.
  • the devices and methods can be used to detect and enrich drug-treated cells (conventional drugs and novel) by their electrical fingerprint to evaluate treatment efficacy.
  • the present inventors have shown that the anti-cancer sphingolipid metabolite sphingosine shifts the cells towards a more benign phenotype while the pro-tumorigenic metabolite sphingosine-1 -phosphate shifts the bioelectrical fingerprint towards a more aggressive phenotype (Salmanzadeh et al., Integr Biol (Camb). 2013 June; 5(6): 843-852).
  • the devices and methods can be used to evaluate the efficacy of conventional drugs and drug combinations and evaluate new compounds/drugs/treatments.
  • the devices and methods can be used to evaluate the sensitivity or resistance of a cancer cell to any chemotherapeutic drug or any anti-cancer agent.
  • agents that cancer cells can be evaluated for resistance or sensitivity include Abiraterone Acetate, ABITREXATE (Methotrexate), ABRAXANE (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ADCETRIS (Brentuximab Vedotin), Ado-Trastuzumab Emtansine, ADRIAMYCIN (Doxorubicin Hydrochloride), ADRUCIL (Fluorouracil), Afatinib Dimaleate, AFINITOR (Everolimus), ALDARA (Imiquimod), Aldesleukin, Alemtuzumab, ALIMTA (Pemetrexed Disodium), ALOXI (Palonosetron Hydrochloride), AMBOCHLORIN (Chlorambucil), AMBOCLOR
  • devices and methods of this disclosure can provide viable, enriched cell sub-populations from heterogeneous tumors (biopsies, needle biopsies, body fluids etc.) for further analyses by off-chip studies to support personalized treatment decisions and efficacy control, i.e., genomic/mutational analyses, molecular and biochemical analyses for biomarker development, for drug screening, etc. to direct ongoing and further treatment.
  • heterogeneous tumors biopsies, needle biopsies, body fluids etc.
  • off-chip studies to support personalized treatment decisions and efficacy control, i.e., genomic/mutational analyses, molecular and biochemical analyses for biomarker development, for drug screening, etc. to direct ongoing and further treatment.
  • the devices and methods can detect and enrich cancer stem or tumor-initiating cells by their bioelectrical fingerprint independent of specific genotype and can detect and enrich tumor cells from mixed biopsy or bodily fluid populations. Further, the devices and methods can detect and isolate different tumor stages for early detection and further analyses. Additional benefits of the devices and methods will be apparent to a skilled artisan.
  • Embodiments of the devices and methods can monitor the change of a cancer cell to more aggressive/less aggressive by their DEP response and monitor subsequent cultured cells to see if there is a shift in their drug response using DEP.
  • DEP DEP
  • cDEP cDEP
  • iDEP iDEP
  • the devices can operate in parallel or in series with other similar devices to enhance selectivity and throughput. Even further, a recirculation channel that allows for serial enrichment by multiple runs from the original sample volume can also be used. Further, embodiments also allow the methods to be run in reverse resulting in negative enrichment.
  • Embodiments of the present disclosure address the question of how can the physical properties of tumors, such as a cell's electrical, optical or mechanical properties, be used to provide earlier or more reliable cancer detection, diagnosis, prognosis, or monitoring of drug response or tumor recurrence.
  • embodiments of this disclosure monitor changes in the electrical signatures of cells as they develop chemotherapeutic resistance or revert to a less aggressive stage.
  • embodiments of this disclosure develop tools to isolate naturally occurring cells from tumor samples that exhibit, by exploiting differences in electrical properties.
  • embodiments of the present disclosure incorporate dielectrophoresis (DEP) and electrorotation (ROT)-based techniques, which manipulate cell motion based on their distinct bioelectrical signatures.
  • Embodiments of methods of the present disclosure can use a novel method developed by the present inventors, contactless dielectrophoresis (cDEP).
  • cDEP is ideally suited for in vitro studies because of the higher throughput compared to ROT or traditional DEP devices, and because it maintains the sterility of the sample in an isolate-and-culture platform, allowing for off-chip analysis.
  • embodiments of methods of the present disclosure utilize the electrical properties of tumors to identify the onset of drug- resistance and to isolate purified populations of chemotherapeutic resistant cells for in vitro analysis.
  • Dr. Davalos' team has invented a novel technique for cell characterization and separation known as contactless dielectrophoresis (cDEP) [20A, 21 A] that overcomes conventional limitations by providing the spatially non-uniform electric field required for DEP while avoiding direct contact between electrodes and biological sample. This method maintains the sterility of the sample and allows for an isolate-and- culture platform or off-chip analysis.
  • Dr. Roberts in collaboration with Dr. Schmelz's group, has developed and characterized a syngeneic mouse cell model for progressive ovarian cancer [22A].
  • Mouse Ovarian Surface Epithelial (MOSE) cells of distinct phenotypes exhibit changes in their gene expression levels and cellular architecture also reported in the human disease [23A] that are associated with stage-specific changes in their biomechanical [24A] and bioelectrical properties [6A].
  • MOSE Mouse Ovarian Surface Epithelial
  • Aspect 1 Characterize the bioelectrical signature of cells, for example cancer cells, associated with drug resistance.
  • Methods of the present disclosure can be used to determine the bioelectrical properties of different cells, such as drug-resistant and normal cells, using dielectrophoresis-based microfluidic devices.
  • cells can be derived from the inventors' syngeneic murine ovarian surface epithelial (MOSE) model. Samples of resistant and normal MOSE populations can be individually interrogated by DEP, such as cDEP and ROT, microdevices to quantify characteristic properties for each population.
  • DEP such as cDEP and ROT
  • Aspect 2 Validate the bioelectrical signature of drug-resistant cancer cells in vivo for comparative assessment of their tumor-initiating potential.
  • Cells harvested from in vivo derived solid tumors and ascites can be selectively sorted based on the bioelectrical signatures defined in Aspect 1 , using DEP, such as cDEP, to isolate putative drug resistance populations.
  • DEP such as cDEP
  • specialized cDEP microdevices can be be designed via computational modeling to selectively separate drug-resistant from normal cancer cell populations. Sorting can be validated in vitro to verify the retention of drug resistance by quantifying response of sorted samples before and after exposure to the relevant chemotherapy.
  • cDEP-identified cells can be implanted into the murine model and tumor outgrowth assessed for validation of the predicted resistance or tumor-initiating potential.
  • Embodiments of methods of the present disclosure take advantage of the dielectrophoretic response of live cells to extract unique biophysical properties that allows for identification and differentiation of drug-resistant, tumor-initiating and drug- sensitive cancer cell types. This mapping of bioelectrical properties of chemotherapeutic-resistant cancer cells to disease stages of non-treated cells can allow for a new, rapid method for determining drug efficacy.
  • Methods of the present disclosure can be used to address one or more of the following: Acquisition of drug resistance and cancer stem-cell likeness or tumor- initiating capacity corresponds with unique bioelectrical signatures of cancer cells that can be utilized to 1 ) predict drug responsiveness or drug resistance and 2) to determine the cancer stem-cell composition and metastatic potential of solid tumors.
  • the bioelectrical signature can serve as an important index of drug responsiveness or risk of tumor recurrence.
  • the inventors have demonstrated that tumor initiating cells can be isolated from normal tumor/cancer cells using DEP [37A]. Therefore, the technique for isolating cells based on drug resistance can also be applied to tumor initiating cells (cancer stem cells) and can be used to identify the optimal drug for treating a patient or to optimize a treatment regimen for a patient.
  • methods of the present disclosure open the door for further development of a rapid biophysics-based method for predicting personalized patient response to chemotherapeutics. Mapping specific drug-resistance conditions to distinct bioelectrical signatures will translate to more reliable cancer diagnosis and drug- response monitoring.
  • Methods of the present disclosure are novel because, to the inventors' knowledge, the bioelectrical signatures of drug-resistant ovarian cancer cells independent of their genotype and their utilization for detection, treatment decisions and control have not been explored previously.
  • the present methods use contactless dielectrophoresis that in contrast to other dielectrophoretic methods support sterile high-throughput cell sorting and characterization, enabling sufficient cell samples for analysis.
  • cDEP technology is rapid and amenable to mass production, making it viable for widespread use.
  • Embodiments of this disclosure envision a form of personalized medicine in which a patient's peritoneal or solid tumor derived cells are assessed at time of presentation for their drug-resistant pre- inclinations in order to personalize their treatment regimen.
  • the cell model utilized here is also novel, as it is a syngeneic mouse ovarian cancer line representing progressive ovarian cancer (not available as human model) that can be used to validate results in vivo in the specific tumor microenvironments.
  • bioelectrical properties of drug-resistant and non-resistant cancer cells differ by their dielectrophoretic responses, stemming from differences in cytoplasm ion content, nucleus-to-cytoplasm ratio, cytoskeleton structure, and membrane morphology and composition and other cell traits (size, morphology, internal organelles, etc.). These physical differences lead to distinctions in cytoplasm conductivity and specific membrane capacitance.
  • Methods of the present disclosure have the potential to define the specific bioelectrical fingerprint of drug resistant and stem-like ovarian cancer cells using the inventors well-characterized progressive mouse ovarian cancer cell system which now includes the tumor-initiating populations and/or the cancer stem-like populations that are derived from the different stages of cancer progression (early, intermediate and highly aggressive, poorly differentiated metastatic variants).
  • the benchmarks for confirmation of their unique bioelectrical signatures will be based on their aggressiveness, their drug resistance/sensitivity profiles, tumor-initiating capacity, and metastatic potential: 1 ) Characterization of the specific bioelectrical fingerprint of drug resistant and stem-like ovarian cancer cells; 2) Bioelectrical profiling of cancer cells as they acquire drug resistance over time; 3) Correlation of the bioelectrical properties with in vivo and in vitro tumorigenicity and metastatic potential; 4) Validation of the results and translation to the human disease by comparison to established human cell lines that are drug responsive or resistant, and primary human cancer cells.
  • bioelectrical signature or “bioelectrical fingerprint” that identifies propensity towards drug resistance and allows for identification of personalized drug cocktail combinations for efficient treatment of cancer, such as advanced stage cancer.
  • DEP DEP
  • cDEP cDEP
  • iDEP iDEP
  • This provides a new focus for cancer treatment and drug-screening efforts from genotypic and biomarker-focused approaches with limited efficiency to characterization and sorting based on the biophysical properties of cancer cells. This will support effective treatment decisions and avoid the use of generic drug cocktails that may delay the use of individually targeted treatments.
  • changes in the bioelectrical signature can be used for treatment control. The characterization of a specific stem-like bioelectrical fingerprint will allow for the immediate determination of the metastatic potential of the tumor and also direct treatment decisions.
  • Embodiments also include a drug:cell profile library which will allow a quick test to determine an optimum regimen.
  • Aspect 1 Characterize the bioelectrical signature of cancer cells associated with drug resistance.
  • the bioelectrical properties of cells can be experimentally determined using DEP-based microfluidic devices. The resulting profiles of resistant cells can be compared to those of TICs from the same line to determine whether resistance confers stem-like or tumor-initiating properties.
  • Methods of the present disclosure can be used to resolve such questions as: 1) Is the crossover frequency altered as cells acquire drug resistance? 2) Is the bioelectrical signature unique to a given drug? (Cisplatin/Carboplatin vs. a taxane such as paclitaxel or docetaxel vs. combinatorial resistance vs gemcitabine etc., also etoposide, and/or doxorubicin or vinorelbine. and 3) Can the cross-over frequency profile predict drug responsiveness prior to the onset of chemotherapy? Meaning can the inventors predetermine which combinatorial drug regimen will be efficacious prior to treatment?
  • Bioelectrical characterization of MOSE cells The bioelectrical signature of cells can be determined by using DEP and electrorotation (ROT), label-free techniques for characterizing, detecting, and isolating cells based on their intrinsic biophysical properties [18A, 19A, 16A, 26A, 27A, 28A, 29A, 30A, 31 A].
  • DEP is the motion of polarized particles in a non-uniform electric field toward the high (positive DEP) or low (negative DEP) gradients of electric field.
  • ROT is the rotation of a cell or particle influenced by a rotating electric field [32A, 33A].
  • the time-average DEP force can be modeled as DEP , m J J !
  • CM Clausius-Mossotti
  • the CM factor is given by ( £ P ⁇ £ m)/ ( E p + 2 m ) j w ere € P is the cell's complex electrical permittivity.
  • ⁇ w is a function of the frequency of the electric field, in addition to morphological and electrical properties of the cell.
  • ISO is the frequency at which the DEP force changes sign.
  • ROT torque depends on the imaginary part, and has its peak close to fss .
  • cDEP has been used in the inventors previous studies to demonstrate that MOSE cells can be differentiated by their bioelectrical crossover frequencies [6A], which were also distinctly different from stromal and immune cells [7A], indicating that cDEP can successfully be used to identify and differentiate ovarian cancer cells from peritoneal cells.
  • bioelectrical crossover frequencies [6A]
  • stromal and immune cells [7A]
  • cDEP can successfully be used to identify and differentiate ovarian cancer cells from peritoneal cells.
  • sphingolipid metabolites with anti-tumor properties or pro-tumorigenic activities differentially modulate the bioelectrical characteristics of late stage MOSE (MOSE-L) cells towards a profile similar to that of benign MOSE E cells. This was associated with a shift of the specific membrane capacitance of MOSE-L cells towards that of MOSE-E cells [8A].
  • both cell lines display unique crossover frequencies that allow for their identification and differentiation from the MOSE-L parental cells. Although preliminary, these data strongly support that the crossover frequency can be employed to detect and characterize cancer cells based on aggressiveness as well as drug responsiveness and tumor-initiating capacity [37A].
  • chemotherapeutic drugs cisplatin/carboplatin, paclitaxel, docetaxel, either alone and in combination (both compound groups are used in standard chemotherapy), vs gemcitabine, etoposide, and daunorubicin/adriamycin or vinca alkaloids such as vincristine, vinorelbine or combinations thereof as representatives for commonly used compounds in standard ovarian cancer care, and experimental chemotherapeutics.
  • additional drug resistant variants can be established using standard cell culture passaging of MOSE-L, in the presence of step-wise increasing doses of chemotherapeutic agents.
  • bioelectrical signature as a function of time post-treatment.
  • the direct correlation with dosing and modulation of bioelectrical signature can be determined. This will allow for further refining of the bioelectrical parameters as well as confirm whether there is an early bioelectrical signature that predicts emergence of drug resistance. This may prove useful in identifying subpopulations of cancer cells that display a propensity for emergence of drug resistant variants.
  • FIG. 1 is graph showing crossover frequency for MOSE cells.
  • the FFL stem cell electrical signature increases compared to the MOSE-L parent stage cells.
  • the bioelectrical signatures of three human ovarian cancer cell lines, A2780 (derived from solid tumor tissue), SKOV3 and OV90 (both derived from malignant ascites) as they acquire drug resistance over time can be compared.
  • A2780 derived from solid tumor tissue
  • SKOV3 and OV90 both derived from malignant ascites
  • the uniqueness of the drug-specific bioelectrical signature can be confirmed and allow for the validation of signatures obtained with the MOSE-L drug resistant variants, which may provide invaluable insights towards a personalized screening approach to optimize which drug cocktail may be most beneficial for a given patient. It is believed that the acquisition of drug resistance confers cancer stem-like or tumor-initiating properties to the cells.
  • the bioelectrical signature can be used to define the TIC population of cells derived from different stages of neoplastic progression as a metric for metastatic potential and as such can provide insight into the fundamental relationship between drug-resistance and stem-like properties.
  • a silicon master stamp can be fabricated on a silicon substrate using Deep Reactive Ion Etching (DRIE) process.
  • DRIE Deep Reactive Ion Etching
  • the scalloping effect, sidewall roughness due to the DRIE etching method, can be removed by a 5min wet etching using TMAH 25% at 70 °C.
  • the liquid phase PDMS made by mixing the monomers and the curing agent in a 10:1 ratio (Sylgrad 184, Dow Corning), can be poured onto the silicon master and can cure for 45min at 100°C.
  • the cured PDMS can be removed and fluidic connections can be punched through with blunt needles.
  • the PDMS replica can be bonded to clean glass slides after treating with air plasma for 2 min.
  • ROT devices can be based on quadripolar electrodes with gold electrodes deposited on glass using Pressure Vapor Deposition and a titanium seed layer [38A]. These can also make these as cDEP devices or fluid electrodes.
  • AC fields can be applied to the microdevices using a combination of a function generator a wideband power amplifier and HV transformer to provide voltage needed.
  • a syringe pump can used to drive samples through the microfluidic chips.
  • An inverted or other style microscope (DMI 6000B, Leica) including digital media capture devices or those with specialized filters attached can be used for monitoring cells in the main channel and the dielectrophoretic behavior of the cells can be recorded. All electrical devices (function generator, oscilloscope, syringe pump, microscope) can be connected to single computer allowing for control and data acquisition.
  • Microdevices can be placed in a vacuum jar for at least 30min before running the experiments to reduce priming issues.
  • the electrode channels and main microchannel can be primed with PBS and then filled with respective fluids: high conductivity electrode and cell suspension mixture.
  • DEP signature Determination of DEP signature.
  • the inventors can measure the crossover frequencies and the ROT spectra using the devices described above, mounted on an inverted microscope equipped with a camera for recording videos of cell motion in response to the field.
  • Cells can be suspended in a low conductivity solution [39A] and placed in the center of the electrodes.
  • the AC signal can be programmed to sweep across frequencies in sync with video recording.
  • ROT AC fields with 90° offsets can be generated by in-house electronics utilizing a THS3092 board, ultimately capable of supplying 7.5 Vpp at 50 Hz - 5 MHz.
  • MATLAB can be used for image processing of the video from each experiment. From cDEP experiments the CM can be determined as the frequency at which cells experience no force.
  • electrorotation experiments the inventors can use a MATLAB script to determine when maximal angular velocity occurs. The inventors can fit CM theoretical curves to all measured data and determine changes in bioelectrical properties of cells.
  • Imaging cells to validate biophysical properties SEM or other microscopy such as confocal microscopy can be used to observe the surface roughness, and immune-fluorescence or confocal microscopy to determine the cytoskeletal structure and membrane morphology.
  • FIG. 2 is a schematic diagram showing a cDEP Device Schematic.
  • FIGS. 3A and 3B show MOSE cells experiencing negative and positive DEP, respectively.
  • Statistical Analysis can be used to determine if the gathered data of different cells are significantly different.
  • Embodiment can be measured and it can be determined how biophysical parameters (e.g., cytoplasm conductivity, membrane permittivity) of cells resistant to chemotherapeutics differ from those of normal cancer cells.
  • biophysical parameters e.g., cytoplasm conductivity, membrane permittivity
  • the bioelectrical signature of drug- resistant cancer cells in vivo for comparative assessment of their tumor-initiating potential can be validated.
  • the bioelectrical signature may or may not be maintained during in vivo dissemination of cancer. More specifically, in some circumstances, ascites-associated cancer cells differ from solid tumor associated cancer cells with respect to their bioelectrical signature.
  • an objective is to employ bioelectrical profiling to predict drug responsiveness at time of presentation.
  • the profiling and sorting protocol Prior to in vivo assessment of the bioelectrical signature of tumor cells, the profiling and sorting protocol, using heterogeneous populations of cells, including the different drug resistant variants, TICs, and fully differentiated parental cells can be used.
  • Cell sorting can be performed at frequency and voltage where subpopulations of cells differ the most.
  • continuous sorting mode mixed cell suspensions can be pumped in the channel, focused to narrow stream, and deflected towards appropriate outlets by DEP force.
  • batch sorting mode cells can be pumped in the channel and one type of cells can be trapped in the regions of high electric field, while other cells can continue to outlet. Untrapped cells can be collected from outlet, trapped cells can be then released from the channel, and collected.
  • the sorted and enriched populations can be subsequently validated for their putative phenotypes as described in more detail below.
  • putative drug-sensitive populations should still exhibit sensitivity to all or selective drugs
  • the putative drug-resistant subsets can be verified by the retention of their drug resistance profile (IC50 assays), and the putative TICs can be confirmed by their enhanced capacity for clonal growth under stem-like growth conditions in serum- free media (spheroid assay) and further defined by their TIC frequency (aka: c-SCF) as determined by extreme limiting dilution analysis [41 A].
  • the putative non-TIC/drug- sensitive subset populations can be used as a negative control for validation of c-SCF.
  • it can be determined whether the drug resistant phenotype and the TIC populations are identical or unique with respect to bioelectrical properties.
  • the TIC population may represent a unique subset population residing within each drug resistant population.
  • Tissue and peritoneal serous fluid harvest can be performed as previously described by the Roberts and Schmelz laboratories [42A]. Briefly, collected ascites cells and tumor tissue (omentum, diaphragm, mesentery) can be disrupted by mild collagenase/hyaluronidase digestion and filtered through 80 and 20 ⁇ cell strainers to obtain single cell suspensions prior to bioelectrical profiling. Cell suspensions can be characterized with cDEP devices in similar fashion. As the MOSE-L/FFL-EGFPTICv represents a tumor- initiating variant, the inventors fully anticipate that upon in vitro propagation, both TIC and more differentiated non-TIC phenotypes can be evident and realized by cDEP.
  • the inventors can further optimize this device to reach the high level of sensitivity and selectivity required to select drug-resistant and/or tumor-initiating cells from a heterogeneous suspension of tumor-associated cells by iteratively re-designing and computationally modeling constrictions in the sample channel in order to have the same field gradient across the whole cross-section of the channel such that cells can be exposed to the same amount of DEP force.
  • the inventors expect that this can increase separation resolution.
  • the investigators can choose to use an number of microfluidic techniques detailed in literature including hydrodynamic focusing.
  • the inventors can focus the initial stream of cell suspension by DEP with another pair of electrodes which causes negative DEP to all cells.
  • the inventors can build electronics capable of independently generating two HV sine signals and design new cDEP devices featuring multiple electrode channels in succession or at divergent axes.
  • the inventors can optimize shape of the channels and throughput to ensure sufficient viable cells out of the device for spheroid formation and in vivo studies. This would yield cDEP devices which employ a multi-frequency cascading platform with multiple outlets to search for subpopulations of resistant cells.
  • a multi-frequency strategy can be employed for conventional DEP cell separation [46A].
  • cells can be separated in sequential steps, such as two or more sequential steps, each operating at two simultaneous frequencies to assess whether there are subpopulations of resistant cells.
  • the heterogeneity of mouse-derived cells may perturb characterization of subpopulations of the cancer cells.
  • the cells can be separated with cDEP devices prior to characterization and then the bioelectrical signature of the separated cells can be measured.
  • Embodiments of this disclosure provide a dielectrophoresis device having a sample channel which is separated by physical barriers from electrode channels which receive electrodes.
  • the electrodes provide an electric current to the electrode channels, which creates a non-uniform electric field in the sample channel, allowing for the separation and isolation of particles in the sample. As the electrodes are not in contact with the sample, electrode fouling is avoided and sample integrity is better maintained.
  • Additional embodiments of this disclosure provide a dielectrophoresis device having a sample channel which is separated by physical barriers from electrode channels which receive electrodes, whereby the sample channel and electrode channels are formed in a single substrate layer and whereby the physical barriers are formed by the substrate itself.
  • Additional embodiments of this disclosure provide a dielectrophoresis device having a channel for receiving a sample in a first substrate layer, a first electrode channel and a second electrode channel for receiving electrodes in a second substrate layer and an insulation barrier between the first substrate layer and the second substrate layer.
  • Additional embodiments of this disclosure provide a dielectrophoresis device having a first electrode channel for conducting an electric current in a first substrate layer, a channel for receiving a sample in a second substrate layer and a second electrode channel for conducting an electric current in a third substrate layer.
  • the device also has a first insulation barrier between the first substrate layer and the second substrate layer and a second insulation barrier between the second substrate layer and the third substrate layer, preventing the sample from coming in contact with the electrodes.
  • Ovarian cancer the most frequent cause of death from gynecological malignancies in women and the fifth leading cause of death from cancer in women [1 , 2] is a genetically and histologically heterogeneous disease. The lack of common genetic markers hinders both cancer detection at earlier stages and the development of successful treatment options.
  • DEP dielectrophoresis
  • DEP has been successfully used for drug screening applications [10] to distinguish between multidrug-resistant and sensitive cancer cells by their cytoplasmic conductivity [1 1 ,12], and to determine cytoplasm and membrane conductivity of drug-treated red blood cells [13]. Further applications of DEP include cell viability determination [10,14] and investigations of drug-stimulated cell surface roughness increase [15].
  • metallic electrodes are used to create a nonuniform electric field [10-15].
  • contact between electrodes and the sample fluid can in some circumstances create challenges for manipulating biological samples including Joule heating, sample contamination, and bubble formation due to electrolysis.
  • a contactless DEP (cDEP) can be used, which is a microfluidic cell manipulation strategy that eliminates direct contact between electrodes and the sample [16].
  • cDEP a contactless DEP
  • an electric field is generated using electrode channels that are separated from the sample channels by a thin insulating barrier. These electrode channels are filled with a highly conductive fluid and under an alternating current (AC) signal are capacitively coupled to the sample channel [17-20].
  • AC alternating current
  • cDEP has been used to isolate prostate tumor initiating cells from prostate cancer cells [21 ] cancer cells from blood cells [22, 23] viable from dead cells [17] and different stages of breast cancer cell lines [24]. Moreover, cDEP has previously been used to quantify dielectric properties of a syngeneic mouse cell model for progressive ovarian cancer [25]. In this model, isolated primary mouse ovarian surface epithelial (MOSE) cells undergo transformation in vitro and progress to malignant stages [26]. Since human cell lines providing different stages of ovarian cancer derived from one genetic source are not available for study, the MOSE model represents a useful alternative that avoids the potential confounding variable of inter-subject genetic differences.
  • MOSE surface epithelial
  • MOSE cells were categorized into early, intermediate, and late stages of malignancy.
  • An increasingly dysregulated cytoskeleton organization and changes in the expression of cytoskeleton genes and their regulators were observed during neoplastic progression, accompanied by an increase in membrane ruffles and protrusions [26, 27].
  • Cytoskeletal changes were associated with stage-specific changes in cellular biomechanical properties [28].
  • the inventors have recently shown for the first time that the dielectric responses of cells are different in different stages of progression [25].
  • the inventors compared the crossover frequency and membrane capacitance of different stages of MOSE cells, finding that the membrane capacitance was greater in malignant cells compared to benign cells [25].
  • Aggressive MOSE cells also showed different dielectric responses from peritoneal cells, specifically macrophages and fibroblasts [29] indicating that cDEP may be an option for isolating ovarian cells from peritoneal fluid for cancer detection.
  • Sphingolipid metabolites influence membrane biology and as lipid second messengers modulate cellular homeostasis, functions and responses to extracellular stimuli. Sphingolipids are involved in the regulation of cell growth, cell death, migration, angiogenesis, and metabolism, among many other cell functions [35, 36]. Dysregulation in metabolic pathways of sphingolipids can cause progression of some diseases, including cancer [37, 38].
  • the sphingolipid metabolites ceramide (Cer), sphingosine (So), and sphingosine-1 -phosphate (S1 P) can stimulate opposing cellular responses depending upon their relative levels in a cell, forming the so-called sphingolipid rheostat [39, 40].
  • So and Cer are known as cell death-promoting factors leading to apoptosis, inhibition of cell growth, differentiation, migration, and angiogenesis [41 ] and thus could be considered tumor suppressors.
  • Cer has also been associated with inflammation [42] suggesting a tumor promoting effect.
  • S1 P acts to support growth and survival of numerous cell types. As such, it has tumor-promoting effects, including inhibition of apoptosis and stimulation of angiogenesis, cell proliferation, differentiation, and migration [41 , 39]. Elevated levels of S1 P have been reported in human ascites fluid of patients with ovarian cancer [43] and may promote the survival, adherence, and outgrowth of peritoneal metastases.
  • mice [00087] Interestingly, therapies targeting S1 P generation and signaling have led to a decreased tumor formation in mice [44].
  • the inventors have used cDEP to characterize MOSE cells' electrical properties after So and S1 P treatment to compare the effects of exogenous sphingolipid metabolites associated with anti- and pro-cancer effects, respectively.
  • the inventors demonstrate that sphingolipid modulation therapy induced distinct changes in the bioelectrical properties of cancer cells.
  • the treatments were non-toxic, allowing the use of cDEP to discriminate among viable MOSE-derived cancer cells.
  • the inventors report that So treatment correlated with a shift in electrical properties of the aggressive MOSE cells towards a profile reminiscent of more benign stages, whereas S1 P did not significantly impact the electrical properties of either early or late stage MOSE cells.
  • the association of the altered electrical phenotype of the So treated cells with cancer suppression and the potential for use of the electrical phenotype as a marker for treatment efficacy can be explored in future studies.
  • Poff is the effective induced dipole moment of the particle and is the root mean square electric field.
  • ⁇ m is the permittivity of the suspending medium
  • r is the radius of the particle
  • Clausius-Mossotti factor is represented as:
  • the sign of the frequency dependent Clausius-Mossotti factor determines the direction of translational particle movement, either toward a region of high electric field gradient (positive DEP, pDEP) or low electric field gradient (negative DEP, nDEP).
  • Biological particles are more complex than a simple spherical particle, and models of varying complexity have emerged that can approximate a biological particle, such as a cell, with sufficient accuracy.
  • a multi-shell model [45] the membrane of the bioparticles, the nucleus, and even the nucleus membrane can be considered, and parameters can be tailored to a specific cell of interest. For the work presented here, a single shell model that considers the cell's thin lipid membrane and the internal cytoplasm is used. Thus the effective permittivity can be written as:
  • the crossover frequency can be used as a tool to monitor the effect of treatments that physically alter the cell.
  • the crossover frequency can be determined by setting
  • dielectric properties of cells are related to membrane properties [48].
  • ms is the surface conductance of the membrane related to the electrical double layer around the cell, and y/mm is the conductance associated with the transport of ions across the membrane [46, 49].
  • ⁇ 100 kHz the low value of ' 3 fn&m, representing the membrane bulk conductivity, prevents the applied electric field from penetrating the interior of the cell.
  • membrane resistance begins to short-circuit and electric field penetrates inside the cell.
  • Equation (5) can be simplified to the form of [46]:
  • Equation (10) shows that there is an inverse relation between the ratio of crossover frequency to sample conductivity, m m and mem . Also it shows that the electrical properties of cells, such as specific membrane capacitance, can be calculated from their crossover frequency.
  • the single-shell model has been successful for predicting the biophysical properties of cells, it sometimes deviates from the experimental results [50] since the real cellular structure is more complex than that assumed by the single-shell model. For instance, this model assumes cells have a thin and spherical membrane which surrounds a spherical homogeneous interior, and thus does not take into account membrane inhomogeneity and cytoplasm and nuclear structural features [51 ].
  • the microdevice shown in FIG. 5, consists of a straight main channel and parallel fluid electrode channels, each 50 ⁇ in depth.
  • Other devices can also be used including those disclosed in U.S. Patent No. 7,678,256 and U.S. Application Publication Nos. 2010/0224493 and 2012/0085649.
  • the main channel has an inlet and outlet with a series of rounded 'sawtooth' features that constrict the main channel from 500 ⁇ width to 100 ⁇ . These sawtooth features create high electric field gradients in the region where the sample channel is constricted, and the series of features increases the length of time that the cells are exposed to the DEP force.
  • Fluidic electrode channels are separated from the sample channel by 20 ⁇ thick insulating barriers.
  • microdevices can consist of a straight main channel and overlaid interdigitated fluid electrode channels.
  • the main channel has an inlet and outlet and the height and width can be tailored for the desired throughput or application.
  • the overlaid electrode channels consist of curved geometry. These channels are separated from the main channel by a 20 ⁇ m thick insulating layer. The curves taper from the bottom side of the channel to the top side (FIG. 6A, 7A).
  • This geometry can be altered to achieve a desired DEP force given a particular frequency by changing parameters such as the angle of curvature, nominal ratio of electrode width to gap width, and number of electrode pairs in series.
  • the nominal ratio of electrode width to gap width is determined by the distances at the bottom of the channel.
  • FIG. 6A shows a surface plot for computational modeling of
  • the legend (units of Hz) shows the frequency range 100-500 kHz.
  • FIG. 7 A shows a surface plot for computational modeling of
  • FIG. 7B shows a line plot along the x-centerline of the sample channel demonstrating the change in * ' ⁇ ⁇ ⁇ ⁇ *3 ⁇ 4 ⁇ with frequency.
  • the legend shows the frequency value in Hz.
  • a stamp of the microdevice design was made for the use with standard soft lithography techniques.
  • AZ 9260 photoresist (AZ Electronic Materials, Somerville, NJ, USA) was spun onto a clean silicon wafer and exposed to UV light for 60 s through a mask patterned with the device design. The exposed photoresist was removed using AZ 400 K developer (AZ Electronic Materials, Somerville, NJ, USA). Deep Reactive Ion Etching (DRIE) was used to etch microchannels to a depth of 50 ⁇ . Surface roughness on the side walls was removed by 5 minutes wet etching with tetramethylammonium hydroxide (TMAH) 25% at 70 °C. A thin coating of Teflon, which improved the release of the device from the stamp, was deposited using DRIE.
  • DRIE Deep Reactive Ion Etching
  • the devices were fabricated from polydimethylsiloxane (PDMS).
  • PDMS was mixed in a 10:1 ratio of elastomer to curing agent (Sylgard 184, Dow Corning, USA).
  • the liquid-phase PDMS was left under vacuum for 30 minutes to remove air bubbles, and was then poured onto the silicon master stamp and cured for 45 minutes at 100°C.
  • the device was trimmed and fluidic connections were punched in the inlet and outlet of each channel with a 1 .5 mm blunt puncher (Howard Electronic Instruments, USA).
  • the PDMS device and a glass microscope slide were cleaned before treating with air plasma for two minutes and bonding together.
  • MOSE cells were cultured in high glucose DMEM (Sigma Aldrich) supplemented with 4% fetal bovine serum (Atlanta Biologicals), 3.7 g/L NaHC0 3 , and 1 % penicillin/streptomycin (Sigma Aldrich).
  • MOSE-E and MOSE-L cells were treated with 1 .5 ⁇ So or 500 nM S1 P as BSA complexes (BSA, fatty acids-free fraction V, Calbiochem) for three passages, allowing 3-4 days between each passage. These treatments were not toxic to the cells.
  • the cells were harvested by trypsinization, washed and resuspended in DEP buffer (8.5% sucrose [wt/vol], 0.3% glucose [wt/vol], 0.725% RPMI [wt/vol]) [52] to a concentration of 3x 1 06 cells/mL.
  • the cells were stained with Calcein-AM (Molecular Probes Inc., Carlsbad, CA, USA), at a concentration of 2 ⁇ _ dye per ml_ cell suspension.
  • the final cell suspension had an averaged conductivity of 96.97 ⁇ 4.15 S/cm, measured using a conductivity meter (Horiba B-173 Twin Conductivity/Salinity Pocket Testers, Cole-Parmer).
  • the output signal from a function generator (GFG-3015, GW Instek, Taipei, Taiwan) was amplified (Model AL-50 HF- A/VT, West Nyack, NY, USA) to produce output voltages ranging from 0-200VRMS at frequencies between 5 and 70 kHz. Voltage and frequency were monitored using an oscilloscope (TDS-1002B, Tektronics Inc. Beaverton, OR, USA) connected to the output of the function generator.
  • Table 1 presents the values of electrical conductivity and permittivity used in the computational modeling.
  • PBS properties were applied to the fluid electrode channels and DEP buffer properties were used for the sample channel.
  • the electrical properties of PDMS used in the model have been reported by the manufacturer (Sylgard 184, Dow Corning, USA).
  • the viscosity and density of water, 0.001 Pa.s and 1000 kg/m 3 , respectively, were used as the viscosity and density of the sample in the main fluidic channel, given the characteristics of DEP buffer.
  • FIG. 8A illustrates the shear rate inside the sample channel.
  • the inlet velocity was set to 56 ⁇ /s based on the experimental flow rate of 0.005 mL/hr.
  • the outlet boundary was set to no viscous stress (Dirichlet condition for pressure). No slip boundary conditions were applied to the walls of the sample channel. Then, the Navier- Stokes equations were solved for an incompressible laminar flow. Thus, the maximum shear rate is significantly lower than the shear rate threshold (approximately 5000 s-1 ) that can cause cell lysis [53, 54].
  • To model the electric field uniform potentials and ground at the source and sink fluid electrode channels, respectively, were applied as the boundary conditions.
  • the Particle Tracing for Fluid Flow module was used to predict the trajectories of particles at different frequencies. Trajectories of 10 particles with uniform initial position distribution were simulated (FIGS 9A and 9B). Drag and DEP forces were added to the model using velocity and electric fields computed from Laminar Flow and Electric Currents modules. The simulations were based on untreated MOSE-L cell properties. Since DEP and drag forces are both proportional to the size of the cells, the smallest cell radius, 5.85 ⁇ , reported previously [29] was used in the simulations.
  • FIG. 9A and 9B demonstrate cells trajectories at 5 and 20 kHz, respectively. At frequencies less than the crossover frequency, cells experience nDEP, thus they are repelled from higher ⁇ ⁇ RM ⁇ * £RMS) and move towards the bottom half of the sample channel (FIG. 9A). At frequencies higher than the crossover frequency cells experience pDEP, thus they are attracted towards higher v * - « f w ⁇ «* - « / w ⁇ anc
  • FIGS. 10A, 10B and 10C demonstrate cell movement in the sample channel without any applied electric field, and due to applying 200 VRMS and at frequencies lower and higher than the crossover frequency, respectively.
  • ⁇ ⁇ MS ⁇ RMS S muc h greater at the top side of the channel due to the sawtooth features, which induce nonuniformities into the electric field.
  • FIG. 10D-F demonstrate the normalized cells distribution corresponding to no DEP force from FIG. 10A, nDEP from FIG. 10B, and pDEP from FIG. 10C, respectively.
  • FIG. 10D shows the distribution of cells without an applied voltage to verify that the cells were randomly distributed in the absence of an electric field. Cell distributions were normalized by the total number of cells crossing the red line in FIGS 10A-10C to make comparing cells distributions in different experiments possible since the number of cells crossing the line is not exactly equal in all of the experiments.
  • the results presented in FIG. 10B and C are in agreement with the computational modeling of the trajectories of particles at 5 and 20 kHz.
  • FIGS 10A-10F As shown in FIGS 10A-10F cells experience a stronger pDEP force than nDEP and they are focused in a narrower stream at the top side of the channel while experiencing pDEP force than when they experience nDEP force, due to two reasons.
  • hM increases as the applied frequency is increased, and because, pDEP for cells occurs at higher frequencies than nDEP, cells experience a stronger DEP force during pDEP than nDEP.
  • the ratio of crossover frequencies to sample conductivity, m m for untreated, So- treated, and S1 P-treated MOSE-E cells were 1 .96 ⁇ 0.1 6, 2.06 ⁇ 0.18, and 2.00 ⁇ 0.39 MHz- m/S, respectively, which were not statistically different, indicating that exogenous sphingolipids do not affect the crossover frequencies of MOSE-E.
  • m m' of MOSE-L cells were 1 .35 ⁇ 0.07, 1 .94 ⁇ 0.07, and
  • ⁇ can be defined as TM ⁇ nwm ⁇ 0 ; where ⁇ 0 is the membrane capacitance of a smooth cell, r f — Q ⁇ ⁇ / ? ⁇
  • M includes three characteristics of cells when are attached in cell culture flask: flatteningon the culture flask surface, cell elongation and the long dendritic projections, and small features, such as ruffles, folds and microvilli on cell surface [63].
  • MOSE-E cells have well-organized, long, cable-like bundles of actin fibers while MOSE-L cells have a highly disorganized actin and microtubule cytoskeleton [26, 27] critical for the viscoelasticity of the cells [28].
  • Stage-dependent, step-wise changes in gene expression levels during MOSE neoplastic progression have been reported previously by using mouse whole genome microarray and gene ontology analyses [26, 27].
  • DEP force to overcome the drag force can be deflected in a stream at the top of the channel while larger particles that require less ⁇ - L ' for the DEP force to overcome the drag force will be deflected to a stream nearer the bottom of the channel.
  • particles with varying biophysical properties should be sorted into streams based on differential DEP forces experienced across the channel. The inventors suggest that this curved design will allow sorting of multiple particles in a "rainbow"-type fashion, where multiple sorted streams can flow to >2 outlets for collection.
  • So treatment correlates with a partial reversal of the aggressive phenotype of late-stage ovarian cancer cells defined by a shift (decrease) in the membrane specific capacitance of MOSE-L cells towards that observed for less aggressive cells.
  • S1 P increased surface membrane protrusions whereas SL-treated cells overall exhibited a smoother surface.
  • the underlying molecular or structural alterations responsible for the changes in dielectric properties and the response to treatment may be critical for the design of devices for cancer detection and treatment control. This would be an advantage over methods that rely solely upon expressed surface receptors, not only for applications such as cell identification and enrichment but also for targeted treatments. Utilizing cDEP for mapping electrical properties of treated cancer cells to specific disease stages of non-treated cells may allow a new, rapid method for determining drug efficacy and for performing dosage studies.
  • Additional references include: Creekmore, A.L. et al, "Regulation of cytoskeleton organization by sphingosine in a mouse cell model of progressive ovarian cancer,” Biomolecules 3(3): 386-407, 2013; and Babahosseini, H., et al., "Roles of bioactive sphingolipid metabolites in ovarian cancer cell biomechanics,” Conf Proc IEEE Eng Med Biol Soc. 2012; 2012:2436-9.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des dispositifs et des procédés de diélectrophorèse (DEP) qui permettent, par tri cellulaire, d'identifier, isoler, et/ou séparer des cellules d'intérêt sur la base des propriétés électriques et physiques des cellules. En particulier, l'invention concerne des systèmes et des procédés pour manipuler des particules en suspension dans un fluide, par exemple, des cellules, des micro- ou nano-particules, en utilisant leurs signatures électriques. De tels procédés peuvent être conduits en utilisant les procédés de DEP, iDEP, et/ou cDEP (diélectrophorèse sans contact, dans laquelle le contact direct entre les électrodes et l'échantillon est évité). Typiquement, un champ électrique est induit dans un échantillon comprenant les particules et/ou cellules cibles, telles que des cellules cancéreuses, et la distribution spatiale des cellules est mesurée pour identifier une ou plusieurs caractéristiques ou propriétés des cellules cancéreuses. Les caractéristiques identifiées des cellules triées peuvent être utilisées pour déterminer l'efficacité et/ou la résistance à un médicament vis-à-vis des cellules.
PCT/US2014/049296 2013-07-31 2014-07-31 Procédés de diélectrophorèse pour déterminer une propriété d'une pluralité de cellules cancéreuses Ceased WO2015017729A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361860309P 2013-07-31 2013-07-31
US61/860,309 2013-07-31

Publications (1)

Publication Number Publication Date
WO2015017729A1 true WO2015017729A1 (fr) 2015-02-05

Family

ID=52432448

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/049296 Ceased WO2015017729A1 (fr) 2013-07-31 2014-07-31 Procédés de diélectrophorèse pour déterminer une propriété d'une pluralité de cellules cancéreuses

Country Status (1)

Country Link
WO (1) WO2015017729A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017020394A1 (fr) * 2015-08-05 2017-02-09 深圳大学 Procédé de détection pharmacodynamique basé sur un champ de force de diélectrophorèse et système associé
US10322192B2 (en) 2016-03-02 2019-06-18 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
JP2022521367A (ja) * 2019-02-22 2022-04-07 ノボキュア ゲーエムベーハー Ｘｅｌｏｘ又はｆｏｌｆｏｘと組み合わせたｔｔフィールドを用いる胃癌の治療

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080182278A1 (en) * 2006-12-07 2008-07-31 Weissman Irving L Identification and isolation of transitional cell carcinoma stem cells
US20100224493A1 (en) * 2009-03-09 2010-09-09 Virginia Tech Intellectual Properties, Inc. Devices and methods for contactless dielectrophoresis for cell or particle manipulation
US20120224053A1 (en) * 2009-06-17 2012-09-06 Board Of Regents, The University Of Texas System Method and apparatus for quantitative microimaging

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080182278A1 (en) * 2006-12-07 2008-07-31 Weissman Irving L Identification and isolation of transitional cell carcinoma stem cells
US20100224493A1 (en) * 2009-03-09 2010-09-09 Virginia Tech Intellectual Properties, Inc. Devices and methods for contactless dielectrophoresis for cell or particle manipulation
US20120224053A1 (en) * 2009-06-17 2012-09-06 Board Of Regents, The University Of Texas System Method and apparatus for quantitative microimaging

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LABEED, FH ET AL.: "Assessment Of Multidrug Resistance Reversal Using Dielectrophoresis And Flow Cytometry.", BIOPHYSICAL JOURNAL., vol. 85, September 2003 (2003-09-01), pages 2028 - 2034 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017020394A1 (fr) * 2015-08-05 2017-02-09 深圳大学 Procédé de détection pharmacodynamique basé sur un champ de force de diélectrophorèse et système associé
US10322192B2 (en) 2016-03-02 2019-06-18 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
US10548986B2 (en) 2016-03-02 2020-02-04 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
JP2022521367A (ja) * 2019-02-22 2022-04-07 ノボキュア ゲーエムベーハー Ｘｅｌｏｘ又はｆｏｌｆｏｘと組み合わせたｔｔフィールドを用いる胃癌の治療
JP7216829B2 (ja) 2019-02-22 2023-02-01 ノボキュア ゲーエムベーハー Ｘｅｌｏｘ又はｆｏｌｆｏｘと組み合わせたｔｔフィールドを用いる胃癌の治療
JP2023053964A (ja) * 2019-02-22 2023-04-13 ノボキュア ゲーエムベーハー Ｘｅｌｏｘ又はｆｏｌｆｏｘと組み合わせたｔｔフィールドを用いる胃癌の治療
JP7419579B2 (ja) 2019-02-22 2024-01-22 ノボキュア ゲーエムベーハー Ｘｅｌｏｘ又はｆｏｌｆｏｘと組み合わせたｔｔフィールドを用いる胃癌の治療

Similar Documents

Publication Publication Date Title
US20140339088A1 (en) Dielectrophoresis methods for determining a property of a plurality of cancer cells
Lin et al. Circulating tumor cells: biology and clinical significance
Ruzycka et al. Microfluidics for studying metastatic patterns of lung cancer
Low et al. Benchtop technologies for circulating tumor cells separation based on biophysical properties
Qian et al. Capturing cancer: emerging microfluidic technologies for the capture and characterization of circulating tumor cells
Chen et al. Clinical applications of NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells
CN105683745A (zh) 用于目标颗粒的分离、捕获和分子分析的方法和装置
EP2781906B1 (fr) Méthode pour identifier des cellules
Salmanzadeh et al. Sphingolipid metabolites modulate dielectric characteristics of cells in a mouse ovarian cancer progression model
Labib et al. Magnetic ranking cytometry: profiling rare cells at the single-cell level
CN104704368A (zh) 肿瘤细胞分离/纯化方法和其使用方法
WO2017027519A1 (fr) Systèmes et procédés pour isoler des particules cibles et leur utilisation dans des procédés diagnostique, pronostique et thérapeutique
Khamenehfar et al. Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip
Velasco et al. Personalized drug efficacy monitoring chip
WO2015017729A1 (fr) Procédés de diélectrophorèse pour déterminer une propriété d'une pluralité de cellules cancéreuses
Lapizco-Encinas Microscale nonlinear electrokinetics for the analysis of cellular materials in clinical applications: a review
Khamenehfar et al. Microfluidic devices for circulating tumor cells isolation and subsequent analysis
Ryuzaki et al. Rapid discrimination of extracellular vesicles by shape distribution analysis
Wu et al. Microfluidic-based isolation of circulating tumor cells with high-efficiency and high-purity
Li et al. Efficient detection and single-cell extraction of circulating tumor cells in peripheral blood
Jiang et al. Classification of tumor subtypes leveraging constriction‐channel based impedance flow cytometry and optical imaging
Vaghef‐Koodehi et al. Dielectrophoresis in Carcinoma Diagnosis: Recent Developments and Applications
Joshi et al. Advances in microfluidic platforms for tumor cell phenotyping: from bench to bedside
Aghajanloo et al. Biomechanics of circulating cellular and subcellular bioparticles: beyond separation
Datar et al. Significance of Studying Circulating Tumor Cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14831187

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14831187

Country of ref document: EP

Kind code of ref document: A1