(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

PURPOSE: To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. MATERIALS AND METHODS: This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. RESULTS: (89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7). CONCLUSION: We have developed a (89)Zr-oxine complex cell tracking technique for use with PET that is applicable to a broad range of cell types and could be a valuable tool with which to evaluate various cell-based therapies.

Imaging the cellular uptake of tiopronin-modified gold nanoparticles.

Well-dispersed gold nanoparticles (NP) coated with tiopronin were synthesized by X-ray irradiation without reducing agents. High-resolution transmission electron microscopy shows that the average core diameters of the NPs can be systematically controlled by adjusting the tiopronin to Au mole ratio in the reaction. Three methods were used to study the NP uptake by cells: quantitative measurements by inductively coupled plasma mass spectrometry, direct imaging with high lateral resolution transmission electron microscopy and transmission X-ray microscopy. The results confirmed that the NP internalization mostly occurred via endocytosis and concerned the cytoplasm. The particles, in spite of their small sizes, were not found to arrive inside the cell nuclei. The synthesis without reducing agents and solvents increased the biocompatibility as required for potential applications in analysis and biomedicine in general.

[Dosimetry of cell-monolayers in multiwell plates]. / Dosimetrie von Monolayer-Kulturen in Multiwellplatten.

AIM: Irradiation of cells in-vitro with unsealed radionuclides is often carried out in cylindrical multi-well-plates. For calculation of the absorbed dose using the sphere model is common. This model assumes a spherical distribution of activity. However, by physical aspects a dose reduction in the peripheral area of the activity volume is expected and predicted especially for high-energy beta-emitting radionuclides. The impact on cellular dosimetry shall be depicted in this paper. METHODS: The dose-distribution inside a multi-well-plate was calculated by convolving the dose distribution around a point source with a given activity. This was performed for the radionuclides I-131, Re-188 and Y-90 in wells of different sizes. For comparison the sphere dose was also calculated. RESULTS: Depending on the beta-energy differences up to 40% between the mean calculated dose and the mean sphere dose were found, whereby calculated dose was always lower than the sphere model prediction. Furthermore a fall-off was calculated for the bottom-dose compared to dose in the centre. An analytical expression was revealed for the bottom-dose with respect to the filling level for three different wells. CONCLUSION: The shape of geometry and the influence on dose distribution must be considered especially at in-vitro exposure with low energy and short range beta-emitting radionuclides. There could be a great impact for exact dose estimation, which is especially necessary to know for comparison of different irradiation experiments (e.g. different radionuclides, various irradiation geometries or comparison with x-rays).

High frequency ultrasound tissue characterization and acoustic microscopy of intracellular changes.

The objective of this work is to investigate changes in the acoustic properties of cells when exposed to chemotherapy for monitoring treatment response. High frequency ultrasound spectroscopy (10-60 MHz) and scanning acoustic microscopy (0.9 GHz) were performed on HeLa cells (Ackermann et al. 1954, Masters 2002) that were exposed to the chemotherapeutic agent cisplatin. Ultrasonic radio-frequency data were acquired from pellets containing HeLa cells after exposure to cisplatin to induce apoptosis. Scanning acoustic and laser fluorescence microscopy images were recorded from single HeLa cells exposed to the same drug. Data acquisition in both cases was performed at several time points throughout the chemotherapeutic treatment for up to 27 h. In the high frequency ultrasound investigation, normalized power spectra were calculated within a region-of-interest. A 20 MHz transducer (f-number 2.35) and a 40 MHz transducer (f-number 3) were used for the data collection in the high frequency ultrasound experiments. The backscatter coefficients, integrated backscatter coefficients, mid-band fit and spectral slope were computed as a function of treatment time to monitor acoustical property changes during apoptosis. Acoustic attenuation was measured using the spectral substitution technique at all time points. Spectral parameter changes were detected after 12 h of exposure and coincided with the initiation of cell damage as assessed by optical microscopy. Integrated backscatter coefficients increased by over 100% between 0 h and 24 h of treatment, with small changes in the associated attenuation ( approximately 0.1 dB/[MHz cm]). Acoustic microscopy was performed at 0.9 GHz frequency. The cell structure was imaged using staining in laser fluorescence microscopy. All cells showed excellent correspondence between the locations of apoptotic nuclear condensation observed in optical imaging and changes in attenuation contrast in acoustic microscopy images. The time after drug exposure at which such changes occurred in the optical images were coincident with the time of changes detected in the acoustic microscopy images and the high frequency ultrasound experiments.

Effect of ultrasound-activated microbubbles on the cell electrophysiological properties.

New clinical applications of ultrasound contrast microbubbles extend beyond imaging and diagnosis toward therapeutic applications. Cell membrane permeability and the uptake of substances have been shown to be enhanced by microbubbles under ultrasound stimulation. However, the mechanisms of action of ultrasound-activated microbubbles are still unknown. The aim of our study was to examine how microbubbles and ultrasound interact with cells in an attempt to understand the sonoporation mechanism. The ruptured-patch-clamp whole-cell technique was used to measure membrane potential variations of a single cell. SonoVue microbubbles and mammary breast cancer cell line MDA-MB-231 were used. Ultrasound was applied using single-element transducers of 1 MHz. Microbubbles and cells were simultaneously video monitored during ultrasound exposure. Our results showed that, during sonoporation, a marked cell membrane hyperpolarization occurs (n = 6 cells) at negative pressures above 150 kPa, indicating the activation of specific ion channels while the cell and the microbubbles remain viable. The hyperpolarization was sustained for as long as the microbubbles are in a direct contact with the cell and the ultrasound waves are transmitted. Smaller acoustic amplitudes induced only mild hyperpolarization, whereas shutting off the ultrasound brings the cell membrane potential to its resting value. However, ultrasound alone did not affect the cell membrane potential. A similar hyperpolarization of the cell membrane was observed when a mechanical pressure was applied on the cell through a glass probe. In conclusion, the results demonstrate that microbubbles' oscillations under ultrasound activation entail modifications of the electrophysiologic cell activities by triggering the modulation of ionic transports through the plasmic cell membrane. However, only cells in direct contact with the microbubbles are impacted. The mechanisms involved are likely related to activation of specific channels sensitive to mechanical stresses (stretch-activated channels) and possibly nonspecific ion channels.

Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip.

Ultrasonic-standing-wave (USW) technology has potential to become a standard method for gentle and contactless cell handling in microfluidic chips. We investigate the viability of adherent cells exposed to USWs by studying the proliferation rate of recultured cells following ultrasonic trapping and aggregation of low cell numbers in a microfluidic chip. The cells form 2-D aggregates inside the chip and the aggregates are held against a continuous flow of cell culture medium perpendicular to the propagation direction of the standing wave. No deviations in the doubling time from expected values (24 to 48 h) were observed for COS-7 cells held in the trap at acoustic pressure amplitudes up to 0.85 MPa and for times ranging between 30 and 75 min. Thus, the results demonstrate the potential of ultrasonic standing waves as a tool for gentle manipulation of low cell numbers in microfluidic systems.

Ultrasound, contrast agents and biological cells; a simplified model for their interaction during in vitro experiments.

A model based on simplifying assumptions is described for the time course of an in vitro experiment in which a beam of ultrasound passes through a suspension of biological cells and gaseous contrast agents (UCAs). It is assumed that cavitation-related activation events (AEs) occur, during each of which a UCA is destroyed or becomes nonfunctional and, at the same time, nearby cells are lysed or otherwise altered. If the UCAs are highly concentrated, the ultrasound attenuation is high and may significantly affect the action. The number of cells affected by each AE depends on the concentrations of cells and UCAs as well as the concentration ratio.

Noninvasive imaging of protein-protein interactions in living organisms.

Genomic research is expected to generate new types of complex observational data, changing the types of experiments as well as our understanding of biological processes. The investigation and definition of relationships among proteins is essential for understanding the function of each gene and the mechanisms of biological processes that specific genes are involved in. Recently, a study by Paulmurugan et al. demonstrated a tool for in vivo noninvasive imaging of protein-protein interactions and intracellular networks.

Possible temperature effects computed for acoustic microscopy used for living cells.

Imaging of living cells or tissues at a microscopic resolution, where GHz frequencies are used, provides a foundation for many new biological applications. The possible temperature increase causing a destructive influence on the living cells should be then avoided. However, there is no information on possible local temperature increases at these very high frequencies where, due to strongly focused ultrasonic beams, nonlinear propagation effects occur. Acoustic parameters of living cells were assumed to be close to those of water; therefore, the power density of heat sources in a water medium was determined as a basic quantity. Hence, the numerical solution of temperature distributions at the frequency of 1 GHz was computed for high and low powers generated by the transducer equal to 0.32 W and 0.002 W. In the first case, typical nonlinear propagation effects were demonstrated and, in the second one, propagation was almost linear. The focal temperature increase obtained in water equaled 14 degrees C for the highest possible theoretical repetition frequency of fr = 10 MHz and for the thermal insulation at the sapphire lens-water boundary. Simultaneously, the scanning velocity of the tested object was assumed to be incomparably low in respect to the acoustic beam velocity. The maximum temperature increase in water occurred exactly at this boundary, being equal there to 20 degrees C. It was shown that, first of all, the very high absorption of water was significant for the temperature distribution in the investigated region, suppressing the focal temperature peaks. Because the temperature increases are proportional to the repetition frequency, so for example, at its practical value of fr = 0.1 MHz, all temperature increases will be 100 times lower than listed above. For the low transducer power of 0.002 W, the corresponding temperature increases were about 140 times lower than those for the high power of 0.32 W. The presented solutions are devoted mainly to the reflection pulse mode; however, they can be also applied for the transmitting (continuous-wave) mode, as shown in an example. Pressure distributions were computed for the acoustic field of the microscope for the first and higher harmonics. Hence, at the frequency of 1 GHz, the effective focal radius in water measured as the -6-dB amplitude pressure drop was found to be 1,1 microm, and 0.7 microm for the second harmonic, independently of the assumed transducer power. So the width of the beam, scanning the living cells in the focal region, was equal to 2.2 microm at the fundamental frequency of 1 GHz.

A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective.

This selective review of the biological effects of ultrasound presents a synopsis of our current understanding of how cells insonated in vitro are affected by inertial cavitation from the standpoint of physical and chemical mechanisms. The focus of this review is on the physical and chemical mechanisms of action of inertial cavitation which appear to be effective in causing biological effects. There are several fundamental conditions which must be satisfied before cavitation-related bioeffects may arise. First, bubbles must be created and then brought into proximity to cells. Exposure methods are critical in this regard, and simple procedures such as rotation of a vessel containing the cells during exposure can drastically alter the results. Second, once association is achieved between bubbles and cells, the former must interact with the latter to produce a bioeffect. It is not certain that the inertial event is the prime mechanism by which cells are lysed; there is evidence that the turbulence associated with bubble translation may cause lysis. Additionally, there appear to be chemical and other physical mechanisms by which inertial cavitation may affect cells; these include the generation of biologically effective sonochemicals and the apparent emission of ultraviolet (UV) and soft X-rays. The evidence for inertial cavitation occurring within cells is critically reviewed.

A model based upon pseudo regular spacing of cells combined with the randomisation of the nuclei can explain the significant changes in high-frequency ultrasound signals during apoptosis.

Recent ultrasound (US) experiments on packed myeloid leukaemia cells have shown that, at frequencies from 32 to 40 MHz, significant increases of signal amplitude were observed during apoptosis. This paper is an attempt to explain these signal increases based upon a simulation of the backscattered signals from the cells nuclei. The simulation is an expansion of work in which a condensed sample of cells, with fairly regular sizes, could be considered as an imperfect crystal. Thus, destructive interference could occur and this would be observed as a large reduced value of backscattered signals compared with the values obtained from a similar, but random, scattering source. This current paper explores the possibility that simple changes in the nuclei, such as their observed condensation or the small loss of nuclei scatterers from cells, could cause a significant increase in the observed backscattered signals. This model indicates that the greater backscattered signals can be explained by further randomisation of the average positions of the scattering sources in each cell. When these "microechoes" are added together, so that the destructive interference is reduced, a large increase in the signal is predicted. The simplified model strongly suggests that much of observed large increases of the backscattered signals could be simply explained by the randomisation of the position of the condensed nuclei during apoptosis, and the destruction of the nuclei could produce further signal amplitude changes due to disruption of the cloud of backscattered waves.

Ultrasonic spectral parameter characterization of apoptosis.

Ultrasound (US) spectral analysis methods are used to analyze the radiofrequency (RF) data collected from cell pellets exposed to chemotherapeutics that induce apoptosis and other chemicals that induce nuclear transformations. Calibrated backscatter spectra from regions-of-interest (ROI) were analyzed using linear regression techniques to calculate the spectral slope and midband fit. Two f/2 transducers, with operating frequencies of 30 and 34 MHz (relative bandwidths of 93% and 78%, respectively) were used with a custom-made imaging system that enabled the collection of the raw RF data. For apoptotic cells, the spectral slope increased from 0.37 dB/MHz before drug exposure to 0.57 dB/MHz 24 h after, corresponding to a change in effective scatterer radius from 8.7 to 3.2 microm. The midband fit increased in a time-dependent fashion, peaking at 13dB 24 h after exposure. The statistical deviation of the spectral parameters was in close agreement with theoretical predictions. The results provide a framework for using spectral parameter methods to monitor apoptosis in in vitro and in in vivo systems and are being used to guide the design of system and signal analysis parameters.

[Application of atomic force microscopy in cell biology].

The atomic force microscopy(AFM), an important instrument for the study of cell biology, has been used to image the living cells and localize the cell surface receptors under physiological conditions by perfect high resolution. It was also utilized in the investigations of the cytoskeleton, biological process and interactions between cells. The applications of AFM in cell biology have been achieved greatly in recent years and many results present a good promise in biomedical and clinical medical fields.

Influence of cell packing by centrifugation on 40-MHz ultrasound backscatter.

High-frequency ultrasound (HFUS) signals backscattered from RBL-2H3 cell pellets prepared under different centrifugal forces were analyzed to investigate the packing effect of cell aggregates. The measurements were performed in a pulse-echo setup with a 40-MHz transducer. The changes of ultrasound signals from cell pellet in backscattered power, statistical parameter, and pellet thickness were monitored after centrifugation at between 100g and 1600g. Experimental results showed that the HFUS backscattered power from cell pellets was inversely proportional to centrifugal force and increased to a plateau within 1-2h after centrifugation. The initial thickness of cell pellets decreased with higher centrifugal force, but the changes in thickness and time that took to reach a plateau increased at higher centrifugal force. The envelope statistics of backscattered signals with Nakagami distribution indicates that the centrifugal force and elapsed time after centrifugation affected the backscattering characteristics. The present study suggests that centrifugal force and data acquisition time after cell pellet formation should be considered in in vitro cell packing method with centrifugation to emulate the tissue in vivo.

Composites of aminodextran-coated Fe3O4 nanoparticles and graphene oxide for cellular magnetic resonance imaging.

Formation of composites of dextran-coated Fe(3)O(4) nanoparticles (NPs) and graphene oxide (Fe(3)O(4)-GO) and their application as T(2)-weighted contrast agent for efficient cellular magnetic resonance imaging (MRI) are reported. Aminodextran (AMD) was first synthesized by coupling reaction of carboxymethyldextran with butanediamine, which was then chemically conjugated to meso-2,3-dimercaptosuccinnic acid-modified Fe(3)O(4) NPs. Next, the AMD-coated Fe(3)O(4) NPs were anchored onto GO sheets via formation of amide bond in the presence of 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC). It is found that the Fe(3)O(4)-GO composites possess good physiological stability and low cytotoxicity. Prussian Blue staining analysis indicates that the Fe(3)O(4)-GO nanocomposites can be internalized efficiently by HeLa cells, depending on the concentration of the composites incubated with the cells. Furthermore, compared with the isolated Fe(3)O(4) NPs, the Fe(3)O(4)-GO composites show significantly enhanced cellular MRI, being capable of detecting cells at the iron concentration of 5 µg mL(-1) with cell density of 2 × 10(5) cells mL(-1), and at the iron concentration of 20 µg mL(-1) with cell density of 1000 cells mL(-1).

Effects of cell spatial organization and size distribution on ultrasound backscattering.

In ultrasound tissue characterization dealing with cellular aggregates (such as tumors), it can be hypothesized that cell microstructure and spatial distribution dominate the backscatter signal. Effects of spatial organization and size distribution of nuclei in cell aggregates on ultrasound backscatter are examined in this work using 2-D computer simulations. The nuclei embedded in cytoplasm were assumed to be weak scatterers of incident ultrasound waves, and therefore multiple scattering could be neglected. The fluid sphere model was employed to obtain the scattering amplitude for each nucleus and the backscatter echo was generated by summing scattered signals originating from many nuclei. A Monte Carlo algorithm was implemented to generate realizations of cell aggregates. It was found that the integrated backscattering coefficient (IBSC) computed between 10 and 30 MHz increased by about 27 dB for a spatially random distribution of mono-disperse nuclei (radius = 4.5 µm) compared with that of a sample of periodically positioned mono-disperse nuclei. The IBSC also increased by nearly 7 dB (between 10 and 30 MHz) for a spatially random distribution of poly-disperse nuclei (mean radius ± SD = 4.5 ± 1.54 µm) compared with that of a spatially random distribution of mono-disperse nuclei. Two different Gaussian pulses with center frequencies 5 and 25 MHz were employed to study the backscatter envelope statistics. An 80% bandwidth was chosen for each case with approximately 0.32 mm as the full-width at half-maximum (FWHM) for the first pulse and 0.06 mm for the second. The incident beam was approximated as a Gaussian beam (FWHM = 2.11 and 1.05 mm for those pulses, respectively). The backscatter signal envelope histograms generally followed the Rayleigh distribution for mono-disperse and poly-disperse samples. However, for samples with partially ordered nuclei, if the irradiating pulse contained a frequency for which ultrasound wavelength and scatter periodicity became comparable (d ~ λ/2), then the histograms were better fitted by the Nakagami distribution. This study suggests that the shape of an envelope histogram depends upon the periodicity in the spatial organization of scatterers and bandwidth of the ultrasound pulse.

Influence of polymer adjuvants on the ultrasound-mediated transfection of cells in culture.

The purpose of this study was to further understand the mechanisms involved in ultrasound-mediated delivery of DNA (sonoporation); in particular, to understand how a plasmid should be formulated with an ultrasound contrast agent (UCA). Different polymer adjuvant-UCA combinations were formulated, and their impact on in vitro DNA transfection, was determined, under various experimental conditions. When present in the medium surrounding a cell suspension, and in the presence of a plasmid encoding for the green fluorescent protein (GFP), expression following sonoporation was increased by more than 1.5-fold compared to that achieved in control experiments (without the adjuvants). The effects of the adjuvants were not influenced by the nature of the UCA, nor by that of the transfected cells; in contrast, the adjuvant concentrations, their physico-chemical properties, and the manner in which they were used, did have an impact on transfection. Close association of the adjuvants to the UCA inhibited their action, suggesting that these substances must have access to the cell membrane to be effective. Indeed, Pluronic F127 appeared to improve the efficacy of transfection (percentage of GFP-positive cells and cell viability), via fluidization of the cell membrane, perhaps facilitating thereby the formation of transient pores and their re-sealing. The mechanism of action of polyethylene glycols, on the other hand, remains unclear.

Laser-induced bubbles in living cells.

BACKGROUND AND OBJECTIVES: Laser-activated micro- and nano-bubbles (LAB) in cells may be used as universal and sensitive non-toxic probes for measuring functional properties of individual cells. STUDY DESIGN/MATERIALS AND METHODS: Such bubbles can be detected and imaged by microscopy and flow cytometry. LABs in living blood and tumor cells were induced by pulsed (532 nm, 10 nanoseconds) laser radiation and detected by the thermal lens optical method. RESULTS: Registered lifetime and maximal diameter of the studied LABs varied within the ranges of 0.02-10 microseconds and 0.44-100 microm, respectively. LAB parameters, thresholds and probabilities, were found to depend upon the physiological state of cells. Specificity and sensitivity of LAB cytometry were increased due to the use of light-absorbing nanoparticles conjugated to specific monoclonal antibodies. CONCLUSIONS: LAB were found to be the universal phenomena that can be used for sensitive and non-invasive monitoring of any individual cell, intact or nanoparticle-treated.

Positioning, displacement, and localization of cells using ultrasonic forces.

This paper presents a method and a device to position and displace cells. The cells are suspended in a fluid layer trapped between the device and an arbitrary surface such as an object slide or a wafer. The device vibrates at ultrasonic frequencies causing a pressure field in the fluid layer. This pressure field results in a force-field capable of positioning cells. Depending on the way in which the device is excited a 2-D or 3-D force-field can be generated, positioning the cells in lines or points respectively. Furthermore, it is possible to subsequently displace the cells with micrometer accuracy. This has been demonstrated using HL60 and MCF10A cells, and can be achieved without causing damage to the cells.