Mordechai Deutsch

A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells.

The importance of research involving non-adherent cell lines, primary cells and blood cells is generally undisputed. However, the task of investigating the complexity and heterogeneity of these cells calls for their long-run monitoring at a single-cell resolution. Such a capability is currently unavailable without having to use disruptive cell tethering. The present Cell Retainer (CR) concept enables high-content correlative multi-parametric measurements, from the functional to molecular level, of the same living individual non-adherent cells within a population. Thereby, despite extensive long-term bio-manipulations, the cells preserve their identity without tethering. Several exemplary experiments, using a microscope-slide-based version of the CR, are presented, which could not be performed by other state of the art methods.

A polymer microstructure array for the formation, culturing, and high throughput drug screening of breast cancer spheroids.

Multicellular spheroid models have been recognized as superior to monolayer cell cultures in antitumor drug screening, but their commercial adaptation in the pharmaceutical industry has been delayed, primarily due to technological limitations. The current study presents a new spheroid culture platform that addresses these technical restrictions. The new culturing device is based on a multiwell plate equipped with a glass bottom patterned with an array of UV adhesive microchambers. Each microchamber is designed to accommodate a single spheroid. The system facilitates the simultaneous creation and culturing of a large number of spheroids, as well as screening their response to antitumor drugs. The volume of the spheroids is easily controlled by seeding density. The location of each spheroid is preserved in the same microchamber throughout its growth, treatment with soluble agents, and imaging. The growth ratio parameter, a non-intrusive size analysis of the same spheroid before and after exposure to drugs, was found to be a sensitive indicator for the reaction of MCF7 breast cancer spheroids to cytotoxic drugs. This feature helps reveal the heterogeneity within the spheroid population during the formation process and their drug response, and provides an opportunity to detect specific, highly active or drug-resistant spheroid sub-groups. The advantages of this spheroid-based system make it an efficient drug-screening tool that may be valuable to related fields of research and clinical applications.

Nanoparticulate flurbiprofen reduces amyloid-β42 generation in an in vitro blood–brain barrier model

IntroductionThe amyloid-β42 (Aβ42) peptide plays a crucial role in the pathogenesis of Alzheimer’s disease (AD), the most common neurodegenerative disorder affecting the elderly. Over the past years, several approaches and compounds developed for the treatment of AD have failed in clinical studies, likely in part due to their low penetration of the blood–brain barrier (BBB). Since nanotechnology-based strategies offer new possibilities for the delivery of drugs to the brain, this technique is studied intensively for the treatment of AD and other neurological disorders.MethodsThe Aβ42 lowering drug flurbiprofen was embedded in polylactide (PLA) nanoparticles by emulsification-diffusion technique and their potential as drug carriers in an in vitro BBB model was examined. First, the cytotoxic potential of the PLA-flurbiprofen nanoparticles on endothelial cells and the cellular binding and uptake by endothelial cells was studied. Furthermore, the biological activity of the nanoparticulate flurbiprofen on γ-secretase modulation as well as its in vitro release was examined. Furthermore, the protein corona of the nanoparticles was studied as well as their ability to transport flurbiprofen across an in vitro BBB model.ResultsPLA-flurbiprofen nanoparticles were endocytosed by endothelial cells and neither affected the vitality nor barrier function of the endothelial cell monolayer. The exposure of the PLA-flurbiprofen nanoparticles to human plasma occurred in a rapid protein corona formation, resulting in their decoration with bioactive proteins, including apolipoprotein E. Furthermore, luminally administered PLA-flurbiprofen nanoparticles in contrast to free flurbiprofen were able to modulate γ-secretase activity by selectively decreasing Aβ42 levels in the abluminal compartment of the BBB model.ConclusionsIn this study, we were able to show that flurbiprofen can be transported by PLA nanoparticles across an in vitro BBB model and most importantly, the transported flurbiprofen modulated γ-secretase activity by selectively decreasing Aβ42 levels. These results demonstrate that the modification of drugs via embedding in nanoparticles is a promising tool to facilitate drug delivery to the brain, which enables future development for the treatment of neurodegenerative disorders like AD.

Analysis of Enzyme Kinetics in Individual Living Cells Utilizing Fluorescence Intensity and Polarization Measurements

BACKGROUND The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular markers mobility restrictions. RESULTS Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.

Intracellular esterase activity in living cells may distinguish between metastatic and tumor-free lymph nodes

Background One of the major clinical problems in breast cancer detection is the relatively high incidence of occult lymph node metastases undetectable by standard procedures. Since the ascertainment of breast cancer stage determines the following treatment, such a “hypo-diagnosis” leads to inadequate therapy, and hence is detrimental for the outcome and survival of the patients. The purpose of our study was to investigate functional metabolic characteristics of living cells derived from metastatic and tumor-free lymph nodes of breast cancer (BC) patients. Methods Our methodology is based on the ability of living cells to hydrolyze fluorescein diacetate (FDA) by intracellular esterases and on the association of FDA hydrolysis rates with a specific cell status, both in physiological and pathological conditions. Results The present study demonstrates a significant difference in the ability to utilize FDA by lymph node cells derived from metastatic and tumor-free lymph nodes in general average, as well as in the metastatic and tumor-free lymph nodes of individual patients. Cells from metastatic lymph nodes had a higher capacity for FDA hydrolysis, and increased this activity after additional activation by autologous tumor tissue (tt). The association between increased FDA hydrolysis rate and activated T lymphocytes and antigen-presenting cells (APC) was shown. Conclusion The results of the present study may contribute to predicting the risk of involvement of seemingly “tumor-free” axillary lymph nodes in occult metastatic processes, and to reducing false-negative results of axillary examination.

Validation of the SCM-test for the diagnosis of cancer

The basic aspects of the SCM-test are summarized. A derivation of the fluorescence intensity vs time curve is given as well as that of the polarization as a function of time. These curves are found to be in excellent agreement with the experimental results. The test is applied to the lymphocytes of twenty-six healthy individuals and of thirty-three cancer patients. No false negative results were obtained. Seven cancer patients were found to be negative after various intervals from the removal of the tumour. The most important technical difficulties and pitfalls are discussed in order to facilitate the reproduction of the test by other researchers.

Polymer live-cell array for real-time kinetic imaging of immune cells

Direct quantitative experimental investigations of the function of lymphocytes and other immune cells are challenging due to the cell mobility and the complexity of intercellular communications. In order to facilitate such investigations, an in vitro system is required that is noninvasive and provides kinetic data on cellular responses to challenges such as drug treatments. The present work reports the development of a disposable, inexpensive polymer-made device, the Polymer Live Cell Array (PLCA), for real-time, kinetic analysis of immune cells. The PLCA proved to be optically and biologically compatible, thus individual immune cells can be observed and treated independently without being tethered. The cells share a common space which facilitates cellular communications via secreted molecules or via direct intercellular interactions. These properties facilitate real-time, non-intrusive, repeated measurements of immune cells under multiple experimental treatments.

Tracing apoptosis and stimulation in individual cells by fluorescence intensity and anisotropy decay.

Presented is the use of fluorescence lifetime (FLT), anisotropy decay, and associated parameters as differential indicators of cellular activity. A specially designed combination of a frequency mode based time resolved microscope and a picoliter well-per-cell array have been used to perform temporal measurements in individual cells under various biological conditions. Two biological models have been examined: mitogenic activation of peripheral blood mononuclear cells (PBMC) and induction of programmed cell death (apoptosis) in Jurkat T cells (JTC). The FLT of fluorescein stained PBMC was found to increase from 4+/-0.02 to 4.5+/-0.025 ns due to mitogenic activation, whereas during apoptosis in fluorescein stained JTC, the FLT remained constant. Notably, the rotational correlation times changed in both models: decreased in PBMC from 2.5+/-0.08 to 2+/-0.1 ns, and increased in JTC from 2.1+/-0.07 to 3.3+/-0.09 ns. FLT and rotational correlation time were used to calculate the steady state fluorescence anisotropy (FA) which was compared to directly measured FA values. The present study suggests that in addition to bioindication, the said parameters can provide valuable information about cellular mechanisms that may involve complex molecular diffusion dynamics, as well as information about structural changes that a cellular fluorophore undergoes in the course of cell activation.

The immunosuppressive effect of methotrexate in active rheumatoid arthritis patients vs. its stimulatory effect in nonactive patients, as indicated by cytometric measurements of CD4+ T cell subpopulations.

This cytometric study assesses the effects of methotrexate (MTX) on the expanded CD4 + lymphocyte population in active and nonactive rheumatoid arthritis (RA) patients. In the active patients, MTX was found to reduce the predominant CD4 + CD28+ subpopulation (by 30%), and the minor subpopulation of CD4 + CD28− (by 34%). The incidence of CD25 phenotype was downregulated by 15%. These reductions can be attributed to immunosuppression through apoptosis, which was demonstrated by MTX‐induced fluorescein diacetate (FDA) hyperpolarization (an established indicator of early apoptosis). In contrast, in nonactive RA patients, the major CD4 + CD28+ subpopulation of small lymphocytes appeared to be activated by MTX, subsequently transforming into a major hyperblast population, whereas the minor CD4 + CD28− subpopulation was not affected by MTX treatment. The activation by MTX in this group of patients is evidenced by MTX‐induced FDA depolarization (an indicator of early activation). Thus, MTX immunosuppressive effect on CD4 + subsets was found in active patients, whereas immunostimulation by MTX was shown in non‐active patients. The found discriminative effect of MTX may suggest a higher effectiveness of low‐dose MTX therapy in active RA patients.

Influence of fluorescence anisotropy on fluorescence intensity and lifetime measurement: theory, Simulations and experiments

The significance of fluorescence anisotropy in fluorescence intensity and lifetime measurements, and erroneous measurements and interpretations resulting from its disregard, are thoroughly discussed, formulated and quantified. In all fluorescence-related measurements-including excitation and emission spectra, relative fluorescence intensity (FI), fluorescenc life time (FLT), fluorescence resonance energy transfer (FRET), etc., with the exception of fluorescence polarization and anisotropy-it is generally true that the higher the fluorescence anisotropy, the greater the distortion of fluorescence measurements. Quantifiable distortions occur when fluorescence measurements are conducted without considering the influence of fluorescence anisotropy. Here, this influence is described by numerous newly developed mathematical expressions which are simulated and experimentally confirmed utilizing single and binary fluorescent solutions of fluorophores with different spectroscopic characteristics. A marked agreement is shown between the theory and experimental data, clearly indicating the legitimacy of the physical suppositions and the mathematical expressions presented in this paper. Practical and instructive implications are discussed. The following findings are of special applicative importance: 1) the existence of an infinite number of couples of Magic Angles; 2) the deviation between two equally fluorescing particles having different fluorescence anisotropies; 3) the relation between the detected fluorescence intensity and anisotropy when measured under various setups of emission and excitation polarizers; 4) the dependence of the artificial normalized steady-state weight of a single-exponentially decaying fluorophore on its fluorescence anisotropy.