Yana Shafran

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.

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.

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.

Monitoring the apoptotic process induced by oxidized low-density lipoprotein in Jurkat T-lymphoblast and U937 monocytic human cell lines

Cell death is a major event in the pathophysiology of atherosclerosis. Oxidized low-density lipoprotein (Ox-LDL), which plays a key role in the atherogenesis, has a powerful cytotoxic effect and causes necrosis or apoptosis of different types of cells. In the present work we studied the mechanism of cell death in two model systems: T lymphocytes and monocytes cell line, exposed to Ox-LDL. Ox-LDL, but not native low-density lipoprotein (LDL), was found to be cytotoxic to both cell types in a dose and time dependent manner. Apoptotic cell deat was analyzed by evaluating cell size, nucleus DNA content and plasma membrane asymmetry. Early cytoplasmic condensation resulting from cell shrinkage was measured by monitoring fluorescence polarization (FP) of fluorescein labeled cells. The redical scavenger superoxide dismutase (SOD), in a time- and dose-dependent manner, reduced the apoptotic effect of Ox-LDL. Hyperpolarization of fluorescein-labeled cells preceded the appearance of phosphatidylserine (PS) on the plasma membrane. This sensitive parameter for early apoptosis detected different cell death kinetics, as well as varying sensitivity to the inhibitory effect of SOD in monocytes and lymphocytes. Such data suggest that reactive oxygen species generation are involved, in Ox-LDL-induced apoptosis and that monocytes are more susceptible to cell death triggered by oxidative stress.

Comparative analysis of cell parameter groups for breast cancer detection

We present a method for the comparative analysis of parameter groups according to their correlation to disease. The theoretical basis of the proposed method is Information Theory and Nonparametric Statistics. Normalized mutual information is used as the measure of correlation between parameters, and statistical conclusions are based on ranking. The fluorescence polarization (FP) parameter is considered as the principal diagnostic characteristic. The FP was measured in fluorescein diacetate (FDA)-stained individual peripheral blood mononuclear cells (PBMC), derived from healthy subjects and breast cancer (BC) patients, under different stimulation conditions: by tumor tissue, the mitogen phytohemagglutinin (PHA) or without the stimulants. The FP parameters were grouped according to their correlation with breast cancer. It was established that the greatest difference between cells of BC patients and healthy subjects is found in the PHA test (parameter P1).

Monitoring of Intracellular Enzyme Kinetic Characteristics of Peripheral Mononuclear Cells in Breast Cancer Patients

A new methodology for the detection of functional response of peripheral blood mononuclear cells against breast cancer (BC) antigens was developed. The method is based on cellular enzymatic activity measurements, using a fluorogenic substrate. We used this method to estimate the kinetic activity of lymphocytes derived from cancer patients and healthy donors. The aim of the study was to determine a possible correlation between the basic characteristics (Km and Vmax) of biochemical enzymatic reactions in live peripheral white mononuclear cells and common clinical-pathological characteristics in BC patients. Our method shows that the enzymatic activity, upon interaction with mitogen or tumor antigens, of the peripheral blood cells in BC patients is different from the enzymatic reactions in healthy individuals. This holds true in the early stages, and the difference persists throughout all of the stages of the disease. This difference is manifested, primarily, by an increase in the Km values after cell incubation with tumor tissue. It was also demonstrated that higher Km values of tumor tissue-activated peripheral blood mononuclear cells are associated with a better prognostic status of the BC patients (lymph node-negative tumors, hormone receptor preservation, and the absence of Her-2/neu protein overexpression). Thus, the present methodology may serve as an additional criterion for prognosis and monitoring, both in BC patients, and in individuals associated with high cancer risk.

Correlative Analyses of Nitric Oxide Generation Rates and Nitric Oxide Synthase Levels in Individual Cells Using a Modular Cell-Retaining Device

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. II: Functional activity of cryopreserved cells

BackgroundThe cryopreservation and thawing processes are known to induce many deleterious effects in cells and might be detrimental to several cell types. There is an inherent variability in cellular responses among cell types and within individual cells of a given population with regard to their ability to endure the freezing and thawing process. The aim of this study was to evaluate the fate of cryopreserved cells within an optical cryo apparatus, the individual-cell-based cryo-chip (i3C), by monitoring several basic cellular functional activities at the resolution of individual cells.ResultsIn the present study, U937 cells underwent the freezing and thawing cycle in the i3C device. Then a panel of vital tests was performed, including the number of dead cells (PI staining), apoptotic rate (Annexin V staining), mitochondrial membrane potential (TMRM staining), cytoplasm membrane integrity and intracellular metabolism (FDA staining), as well as post-thawing cell proliferation assays. Cells that underwent the freezing - thawing cycle in i3C devices exhibited the same functional activity as control cells. Moreover, the combination of the multi-parametric analysis at a single cell resolution and the optical and biological features of the device enable an accurate determination of the functional status of individual cells and subsequent retrieval and utilization of the most valuable cells.ConclusionsThe means and methodologies described here enable the freezing and thawing of spatially identifiable cells, as well as the efficient detection of viable, specific, highly biologically active cells for future applications.

A novel approach for on line monitoring of apoptotic cell shrinkage in individual live lymphocytes.

The apoptotic process occurs asynchronically in most cell populations and its duration is variable. Therefore, the ability to continuously monitor the death process occurring in individual blood cells before, during and following apoptosis induction is crucial in the evaluation of the efficiency of pro- or anti-apoptotic drugs. We applied a kinetic approach by performing real time measurements of individual living cells. This approach is based on an easy and unique method for monitoring intracellular staining reaction, which accompanied early apoptotic cell shrinkage. The intracellular enzymatic reaction rates were determined by taking repeated, sequential measurements of fluorescence intensity of the same individual cells. These rates were found to correlate with the respective radii of the cells under different conditions, and to decrease following apoptosis induction. The ability to remeasure the same cell before and after apoptosis induction enabled the detection of specific individual lymphocytes, which were more susceptible or resistant to pro-apoptotic stimulus.