Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

Rapid real-time recirculating PCR using localized surface plasmon resonance (LSPR) and piezo-electric pumping.

Rapid detection and characterization of pathogens in patients with bloodstream infections (BSIs) is a persistent problem for modern medicine, as current techniques are slow or provide incomplete diagnostic information. Real-time polymerase chain reaction (qPCR) allows specific detection of a wide range of targets and quantification of pathogenic burdens to aid in treatment planning. However, new technological advances are required for a rapid and multiplex implementation of qPCR in clinical applications. In this paper, the feasibility of a novel microfluidic platform for qPCR is presented, integrating highly sensitive, label-free localized surface plasmon resonance (LSPR) imaging of DNA hybridization into a recirculating chip design for real-time analysis. Single target and multiplex detection of DNA target amplification are demonstrated, with a limit of detection of 5 fg µL-1 of E. coli DNA for single target PCR, correlating with approximately 300 bacteria per mL. The results of this study demonstrate the potential of this platform for simultaneous real-time detection of multiple target genes within 15 minutes that could provide live saving benefits in patients with BSIs.

Isolation and concentration of bacteria from blood using microfluidic membraneless dialysis and dielectrophoresis.

A microfluidic system that combines membraneless microfluidic dialysis and dielectrophoresis to achieve label-free isolation and concentration of bacteria from whole blood is presented. Target bacteria and undesired blood cells are discriminated on the basis of their differential susceptibility to permeabilizing agents that alter the dielectrophoretic behavior of blood cells but not bacteria. The combined membraneless microdialysis and dielectrophoresis system isolated 79 ± 3% of Escherichia coli and 78 ± 2% of Staphylococcus aureus spiked into whole blood at a processing rate of 0.6 mL h-1. Collection efficiency was independent of the number of target bacteria up to 105 cells. Quantitative PCR analysis revealed that bacterial 16S rDNA levels were enriched more than 307-fold over human DNA in the fraction recovered from the isolation system compared with the original specimen. These data demonstrate feasibility for an instrument to accelerate the detection and analysis of bacteria in blood by first isolating and concentrating them in a microchamber.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

A High-Voltage SOI CMOS Exciter Chip for a Programmable Fluidic Processor System.

A high-voltage (HV) integrated circuit has been demonstrated to transport fluidic droplet samples on programmable paths across the array of driving electrodes on its hydrophobically coated surface. This exciter chip is the engine for dielectrophoresis (DEP)-based micro-fluidic lab-on-a-chip systems, creating field excitations that inject and move fluidic droplets onto and about the manipulation surface. The architecture of this chip is expandable to arrays of N X N identical HV electrode driver circuits and electrodes. The exciter chip is programmable in several senses. The routes of multiple droplets may be set arbitrarily within the bounds of the electrode array. The electrode excitation waveform voltage amplitude, phase, and frequency may be adjusted based on the system configuration and the signal required to manipulate a particular fluid droplet composition. The voltage amplitude of the electrode excitation waveform can be set from the minimum logic level up to the maximum limit of the breakdown voltage of the fabrication technology. The frequency of the electrode excitation waveform can also be set independently of its voltage, up to a maximum depending upon the type of droplets that must be driven. The exciter chip can be coated and its oxide surface used as the droplet manipulation surface or it can be used with a top-mounted, enclosed fluidic chamber consisting of a variety of materials. The HV capability of the exciter chip allows the generated DEP forces to penetrate into the enclosed chamber region and an adjustable voltage amplitude can accommodate a variety of chamber floor thicknesses. This demonstration exciter chip has a 32 x 32 array of nominally 100 V electrode drivers that are individually programmable at each time point in the procedure to either of two phases: 0deg and 180deg with respect to the reference clock. For this demonstration chip, while operating the electrodes with a 100-V peak-to-peak periodic waveform, the maximum HV electrode waveform frequency is about 200 Hz; and standard 5-V CMOS logic data communication rate is variable up to 250 kHz. This HV demonstration chip is fabricated in a 130-V 1.0-mum SOI CMOS fabrication technology, dissipates a maximum of 1.87 W, and is about 10.4 mm x 8.2 mm.

PROGRAMMABLE DIELECTROPHORETIC µTAS SAMPLE HANDLING.

We present the concept of a general-purpose sample analysis platform (GSAP) based on dielectrophoretic methods. The platform architecture comprises integrated functional blocks that can be programmed to perform a diverse range of analysis steps, including the on-device preparation of real world samples.

Differential analysis of human leukocytes by dielectrophoretic field-flow-fractionation.

The differential analysis of human leukocytes has many important biological and medical applications. In this work, dielectrophoretic field-flow-fractionation (DEP-FFF), a cell-separation technique that exploits the differences in the density and dielectric properties of cells, was used to separate the mixtures of the major human leukocyte subpopulations (T- and B-lymphocytes, monocytes, and granulocytes). The separation was conducted in a thin chamber equipped with an array of microfabricated interdigitated electrodes on the bottom surface, and the separation performance was characterized by on-line flow cytometry. To investigate optimal separation conditions for different leukocyte mixtures, elution fractograms at various DEP field frequencies were obtained for each leukocyte subtype. With appropriately chosen conditions, high separation performance was achieved in separating T- (or B-) lymphocytes from monocytes, T- (or B-) lymphocytes from granulocytes, and monocytes from granulocytes. DEP-FFF does not involve cell-labeling or cell-modification step, and provides a new approach to hematological analysis.

Cell separation by dielectrophoretic field-flow-fractionation.

Dielectrophoretic field-flow-fractionation (DEP-FFF) was applied to several clinically relevant cell separation problems, including the purging of human breast cancer cells from normal T-lymphocytes and from CD34+ hematopoietic stem cells, the separation of the major leukocyte subpopulations, and the enrichment of leukocytes from blood. Cell separations were achieved in a thin chamber equipped with a microfabricated, interdigitated electrode array on its bottom wall that was energized with AC electric signals. Cells were levitated by the balance between DEP and sedimentation forces to different equilibrium heights and were transported at differing velocities and thereby separated when a velocity profile was established in the chamber. This bulk-separation technique adds cell intrinsic dielectric properties to the catalog of physical characteristics that can be applied to cell discrimination. The separation process and performance can be controlled through electronic means. Cell labeling is unnecessary, and separated cells may be cultured and further analyzed. It can be scaled up for routine laboratory cell separation or implemented on a miniaturized scale.

Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion.

The separation and purification of human blood cell subpopulations is an essential step in many biomedical applications. New dielectrophoretic fractionation methods have great potential for cell discrimination and manipulation, both for microscale diagnostic applications and for much larger scale clinical problems. To discover whether human leukocyte subpopulations might be separable by such methods, the dielectric characteristics of the four main leukocyte subpopulations, namely, B- and T-lymphocytes, monocytes, and granulocytes, were measured by electrorotation over the frequency range 1 kHz to 120 MHz. The subpopulations were derived from human peripheral blood by magnetically activated cell sorting (MACS) and sheep erythrocyte rosetting methods, and the quality of cell fractions was checked by flow cytometry. Mean specific membrane capacitance values were calculated from the electrorotation data as 10.5 (+/- 3.1), 12.6 (+/- 3.5), 15.3 (+/- 4.3), and 11.0 (+/- 3.2) mF/m2 for T- and B-lymphocytes, monocytes, and granulocytes, respectively, according to a single-shell dielectric model. In agreement with earlier findings, these values correlated with the richness of the surface morphologies of the different cell types, as revealed by scanning electron microscopy (SEM). The data reveal that dielectrophoretic cell sorters should have the ability to discriminate between, and to separate, leukocyte subpopulations under appropriate conditions.

Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation.

Dielectrophoretic/gravitational field-flow fractionation (DEP/G-FFF) was used to separate cultured human breast cancer MDA-435 cells from normal blood cells mixed together in a sucrose/dextrose medium. An array of microfabricated, interdigitated electrodes of 50 microns widths and spacings, and lining the bottom surface of a thin chamber (0.42 mm H x 25 mm W x 300 mm L), was used to generate DEP forces that levitated the cells. A 10-microL cell mixture sample containing approximately 50,000 cells was introduced into the chamber, and cancerous and normal blood cells were levitated to different heights according to the balance of DEP and gravitational forces. The cells at different heights were transported at different velocities under the influence of a parabolic flow profile that was established in the chamber and were thereby separated. Separation performance depended on the frequency and voltage of the applied DEP field and the fluid-flow rate. It took as little as 5 min to achieve cell separation. An analysis of the DEP/G-FFF results revealed that the separation exploited the difference in dielectric and density properties between cell populations. The DEP/G-FFF technique is potentially applicable to many biological and biomedical problems, especially those related to microfluidic systems.

Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation.

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.

Role of peroxide in AC electrical field exposure effects on friend murine erythroleukemia cells during dielectrophoretic manipulations.

The effects of AC field exposure on the viability and proliferation of mammalian cells under conditions appropriate for their dielectrophoretic manipulation and sorting were investigated using DS19 murine erythroleukemia cells as a model system. The frequency range 100 Hz-10 MHz and medium conductivities of 10 mS/m, 30 mS/m and 56 mS/m were studied for fields generated by applying signals of up to 7V peak to peak (p-p) to a parallel electrode array having equal electrode widths and gaps of 100 micrometer. Between 1 kHz and 10 MHz, cell viability after up to 40 min of field exposure was found to be above 95% and cells were able to proliferate. However, cell growth lag phase was extended with decreasing field frequency and with increasing voltage, medium conductivity and exposure duration. Modified growth behavior was not passed on to the next cell passage, indicating that field exposure did not cause permanent alterations in cell proliferation characteristics. Cell membrane potentials induced by field exposure were calculated and shown to be well below values typically associated with cell damage. Furthermore, medium treated by field exposure and then added to untreated cells produced the same modifications of growth as exposing cells directly, and these modifications occurred only when the electrode polarization voltage exceeded a threshold of approximately 0.4 V p-p. These findings suggested that electrochemical products generated during field exposure were responsible for the changes in cell growth. Finally, it was found that hydrogen peroxide was produced when sugar-containing media were exposed to fields and that normal cell growth could be restored by addition of catalase to the medium, whether or not field exposure occurred in the presence of cells. These results show that AC fields typically used for dielectrophoretic manipulation and sorting of cells do not damage DS19 cells and that cell alterations arising from electrochemical effects can be completely mitigated.

The removal of human breast cancer cells from hematopoietic CD34+ stem cells by dielectrophoretic field-flow-fractionation.

Dielectrophoretic field-flow-fractionation (DEP-FFF) was used to purge human breast cancer MDA-435 cells from hematopoietic CD34+ stem cells. An array of interdigitated microelectrodes lining the bottom surface of a thin chamber was used to generate dielectrophoretic forces that levitated the cell mixture in a fluid flow profile. CD34+ stem cells were levitated higher, were carried faster by the fluid flow, and exited the separation chamber earlier than the cancer cells. Using on-line flow cytometry, efficient separation of the cell mixture was observed in less than 12 min, and CD34+ stem cell fractions with a purity >99.2% were obtained. The method of DEP-FFF is potentially applicable to many biomedical cell separation problems, including microfluidic-scale diagnosis and preparative-scale purification of cell subpopulations.

Separation of polystyrene microbeads using dielectrophoretic/gravitational field-flow-fractionation.

The characterization of a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system using model polystyrene (PS) microbeads is presented. Separations of PS beads of different surface functionalization (COOH and none) and different sizes (6, 10, and 15 microm in diameter) are demonstrated. To investigate the factors influencing separation performance, particle elution times were determined as a function of particle suspension conductivity, fluid flow rate, and applied field frequency and voltage. Experimental data were analyzed using a previously reported theoretical model and good agreement between theory and experiment was found. It was shown that separation of PS beads was based on the differences in their effective dielectric properties. Particles possessing different dielectric properties were positioned at different heights in a fluid-flow profile in a thin chamber by the balance of DEP and gravitational forces, transported at different velocities under the influence of the fluid flow, and thereby separated. To explore hydrodynamic (HD) lift effects, velocities of PS beads were determined as a function of fluid flow rate in the separation chamber when no DEP field was applied. In this case, particle equilibrium height positions were governed solely by the balance of HD lift and gravitational forces. It was concluded that under the experimental conditions reported here, the DEP force was the dominant factor in controlling particle equilibrium height and that HD lift force played little role in DEP/G-FFF operation. Finally, the influence of various experimental parameters on separation performance was discussed for the optimization of DEP/G-FFF.

Introducing dielectrophoresis as a new force field for field-flow fractionation.

We present the principle of cell characterization and separation by dielectrophoretic field-flow fractionation and show preliminary experimental results. The operational device takes the form of a thin chamber in which the bottom wall supports an array of microelectrodes. By applying appropriate AC voltage signals to these electrodes, dielectrophoretic forces are generated to levitate cells suspended in the chamber and to affect their equilibrium heights. A laminar flow profile is established in the chamber so that fluid flows faster with increasing distance from the chamber walls. A cell carried in the flow stream will attain an equilibrium height, and a corresponding velocity, based on the balance of dielectrophoretic, gravitational, and hydrodynamic lift forces it experiences. We describe a theoretical model for this system and show that the cell velocity is a function of the mean fluid velocity, the voltage and frequency of the signals applied to the electrodes, and, most significantly, the cell dielectric properties. The validity of the model is demonstrated with human leukemia (HL-60) cells subjected to a parallel electrode array, and application of the device to separating HL-60 cells from peripheral blood mononuclear cells is shown.

Dielectrophoretic manipulation of cells with spiral electrodes.

Electrokinetic responses of human breast cancer MDA-MB-231 cells were studied in suspensions of conductivities 18, 56, and 160 mS/m on a microelectrode array consisting of four parallel spiral electrode elements energized with phase-quadrature signals of frequencies between 100 Hz and 100 MHz. At low frequencies cells were levitated and transported toward or away from the center of the spiral array, whereas at high frequencies cells were trapped at electrode edges. The frequencies of transition between these characteristic cell behaviors increased with increasing suspension conductivity. Levitation heights and radial velocities were determined simultaneously for individual cells as a function of the applied field magnitude and frequency. Results were compared with theoretical predictions from generalized dielectrophoresis theory applied in conjunction with cell dielectric parameters and simulated electric field distributions corrected for electrode polarization effects. It was shown that the conventional and traveling-wave dielectrophoretic force components dominated cell levitation and radial motion, respectively. Both theoretical predictions and experimental data showed that the cell radial velocity was very sensitive to the field frequency when the in-phase component of the field-induced polarization was close to zero. Applications of spiral electrode arrays, including the isolation of cells of clinical relevance, are discussed.

Dielectrophoretic detection of changes in erythrocyte membranes following malarial infection.

The dielectric properties of normal erythrocytes were compared to those of cells infected with the malarial parasite Plasmodium falciparum. Normal cells provided stable electrorotation spectra which, when analyzed by a single-shelled oblate spheroid dielectric model, gave a specific capacitance value of 12 +/- 1.2 mF/m2 for the plasma membrane, a cytoplasmic permittivity of 57 +/- 5.4 and a cytoplasmic conductivity of 0.52 +/- 0.05 S/m. By contrast, parasitized cells exhibited electrorotation spectra with a time-dependency that suggested significant net ion outflux via the plasma membrane and it was not possible to derive reliable cell parameter values in this case. To overcome this problem, cell membrane dielectric properties were instead determined from dielectrophoretic crossover frequency measurements made as a function of the cell suspending medium conductivity. The crossover frequency for normal cells depended linearly on the suspension conductivity above 20 mS/m and analysis according to the single-shelled oblate spheroid dielectric model yielded values of 11.8 mF/m2 and 271 S/m2, respectively, for the specific capacitance and conductance of the plasma membrane. Unexpectedly, the crossover frequency characteristics of parasitized cells at high suspending medium conductivities were non-linear. This effect was analyzed in terms of possible dependencies of the cell membrane capacitance, conductance or shape on the suspension medium conductivity, and we concluded that variations in the membrane conductance were most likely responsible for the observed non-linearity. According to this model, parasitized cells had a specific membrane capacitance of 9 +/- 2 mF/m2 and a specific membrane conductance of 1130 S/m2 that increased with increasing cell suspending medium conductivity. Such conductivity changes in parasitized cells are discussed in terms of previously observed parasite-associated membrane pores. Finally, we conclude that the large differences between the dielectrophoretic crossover characteristics of normal and parasitized cells should allow straightforward sorting of these cell types by dielectrophoretic methods.

Electrorotation of liposomes: verification of dielectric multi-shell model for cells.

The reliability of multi-shell dielectric models, used to describe the ac electrokinetic behaviour of cells, has been tested by performing electrorotation and dielectrophoretic measurements on unilamellar, oligolamellar, and multilamellar liposomes of diameters ranging from 5 to 24 microm. Fluorescence microscopy, flow cytometry and electron spin resonance were used to characterise the morphology and membranes of the liposomes. The dielectric properties of the various types of liposomes, based on appropriate dielectric shell models, were then analysed using a general purpose, recursive, algorithm. Through simulations, the confidence levels that can be assigned to parameters derived through application of simple shell models are estimated. From this, we confirm that electrorotation data enable accurate determinations to be made of the dielectric properties of the outermost membrane of liposomes, and provide good indications of the level of complexity of the shells and internal compartments. We also demonstrate that, used with sufficient additional information, such as that provided by dielectrophoresis, electrorotation data yields unique solutions for the dielectric parameters of liposome-like particles.

Membrane changes associated with the temperature-sensitive P85gag-mos-dependent transformation of rat kidney cells as determined by dielectrophoresis and electrorotation.

Conventional dielectrophoresis (cDEP) and electrorotation (ROT) measurements have been used to determine the dielectric properties of a clone of normal rat kidney cells, designated 6m2, that exhibits a transformed phenotype at 33 degrees C and a non-transformed phenotype at 39 degrees C. cDEP measurements of the crossover frequencies at which individual 6m2 cells experienced zero cDEP force performed as a function of the conductivity of the suspension medium revealed that, in response to a temperature shift from 33 degrees C to 39 degrees C for 24 h, the mean specific cell membrane capacitance and conductance fell significantly (P < 0.01) from 42.3 (+/-1.3) to 30.3 (+/-2.9) mF/m2 and 743 (+/-422) to 567 (+/-326) S/m2, respectively. ROT analyses demonstrated a similar reduction for the membrane capacitance from 37.2 (+/-7.3) to 27.4 (+/-6.1) mF/m2, and also showed that accompanying changes in the mean internal electrical conductivity and dielectric permittivity of the cells were insignificant. Scanning electron microscopy was used to examine the surface morphology of the cells and, in agreement with our previous reports for leukemia cells, the observed membrane capacitance values correlated closely with the morphological complexity of the cell membrane surface. The observed changes in the membrane dielectric properties are discussed in terms of their biological significance and their relationship to previously-detected changes in cell surface charge.

Electrorotational studies of the cytoplasmic dielectric properties of Friend murine erythroleukaemia cells.

Electrorotation (ROT) spectra of Friend murine erythroleukaemia DS19 cells were measured in the frequency range 10 kHz-100 MHz as a function of suspension osmolality and cell differentiation treatment with hexamethylene bisacetamide (HMBA). A minimization program was employed to curve-fit the measured spectra using a single-shell dielectric model, allowing the derivation of cellular interior conductivity and permittivity (valid for the frequency range 10-100 MHz) and the cytoplasmic membrane capacitance (its dependence on the cell differentiation state and suspension osmolality having been reported earlier). Following HMBA treatment, DS19 cells exhibited a slight increment in average interior permittivity and a decrement in interior conductivity, although the changes were not statistically significant. For both untreated and HMBA treated samples, the average interior conductivity increased and permittivity decreased with increasing suspension osmolality. Of significance was that the average permittivity of cell interiors was larger than that of pure water. The electrorotation spectra of freshly prepared cell nuclei were measured, and the derived nuclear dielectric parameters were employed in numerical simulations to investigate the effects of nuclei on the ROT spectra of intact cells. Other cellular internal structures such as mitochondria were also analysed using theoretical simulations. It was concluded that the derived large permittivity values did not result from cell nuclei or mitochondria, and, instead, we suggest that they may arise from the combined effects of several cytoplasmic organelles.