RESUMO
Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities-pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion.
Assuntos
Técnicas de Cultura de Células/instrumentação , Imagem Molecular/instrumentação , Proteínas/metabolismo , Análise de Célula Única/métodos , Linfócitos T/citologia , Linfócitos T/metabolismo , Fenômenos Fisiológicos Celulares , Células HEK293 , Células HeLa , Humanos , CinéticaRESUMO
Dynamic light scattering analysis has been demonstrated recently to be a promising tool for the assessment of structural changes taking place inside opaque tissue samples. Specifically, quantification of velocity and direction of cellular motion inside spheroids and organoids has attracted much attention as a potent indicator in personalized therapy research. Here, we propose a method for the quantitative extraction of cellular motion, velocity, and direction, by applying a concept of speckle spatial-temporal correlation dynamics. Numerical simulations and experimental results obtained on phantom and biological spheroids are presented.
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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.
Assuntos
Imuno-Histoquímica , Microscopia Confocal , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Linhagem Celular , Fluoresceína/química , Corantes Fluorescentes/química , Glutationa/metabolismo , Humanos , Lisofosfatidilcolinas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
In response to the need for reliable cellular models that reflect complex tumor microenvironmental properties, and enable more precise testing of anti-cancer therapeutics effects on humans, a co-culture platform for in-vitro model that enhances the physiology of breast cancer (BC) microenvironment is presented. A six well imaging plate wherein each macro-well contains several separate compartments was designed. Three-dimensional (3D) cancer spheroids are generated and cultured in the inner compartment which is embossed with an array of nano-liter micro-chambers made of hydrogel. Stromal cells are cultured in the outer chambers. The two cell types are cultured side-by-side, sharing a common space, thus enabling extra-cellular communication via secreted molecules. As proof of concept, a model of BC tumor microenvironment was recapitulated by co-cultivating 3D MCF7 spheroids in the presence of tumor-associated macrophages (TAMs). The presence of TAMs induced an aggressive phenotype by promoting spheroid growth, enhancing survivin expression levels and enabling invasive behavior. Moreover, TAMs influenced the response of BC spheroids to cytotoxic treatment as well as hormonal drug therapy, and enhanced the effects of nitric oxide donor. The platform enables time-lapse imaging and treatment without losing spatial location of the measured spheroids, thereby allowing measurements and analysis at individual-object resolution in an easy and efficient manner.
Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Neoplasias da Mama/tratamento farmacológico , Técnicas de Cocultura , Doxorrubicina/farmacologia , Humanos , Hidrogéis , Células MCF-7 , Macrófagos/efeitos dos fármacos , Modelos Biológicos , Esferoides Celulares/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Tamoxifeno/farmacologia , Triazenos/farmacologia , Microambiente Tumoral , Células U937RESUMO
BACKGROUND: Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient. RESULTS: The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing--thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing--thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior. CONCLUSIONS: The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing--thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.
Assuntos
Sobrevivência Celular , Criopreservação/métodos , Medicina Regenerativa , Congelamento/efeitos adversos , Humanos , Células U937RESUMO
BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: The 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.
Assuntos
Criopreservação/métodos , Anexina A5/metabolismo , Apoptose , Proliferação de Células , Sobrevivência Celular , Criopreservação/instrumentação , Congelamento , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Células U937RESUMO
The development of distant metastases is the major cause of death in breast cancer (BC). In many BC cases, metastases are present in patients with no metastasis-positive lymph nodes (LN). Hence, there is a need to improve prognosis by a better prediction of the nodal status and tumor spread. The current study is designed to develop and utilize new functional characteristics of the cells and microenvironment of BC-draining LN, which may help to improve the estimation of LN metastatic involvement. Innovative devices and methodologies were developed for collecting, transferring, and analyzing LN at an individual-cell resolution. Using these devices, a suspension of living cells were prepared from the LN and processed for various assays, including immunophenotypic analysis, activation status, and invasion activity. The functional profile of tumor-activated LN cells showed an increase in the intracellular enzymatic reaction rate, accompanied by a homogeneous distribution of transferrin receptor as well as by a significant increase in matrix metalloproteinase proteolytic activity. Moreover, the proportion of cells exhibiting such a profile was significantly higher in tumor-containing LN than in tumor-free LN. Thus, the live and postfixation features of LN cells and their microenvironment, correlated with the functional status of the LN, may serve to improve their predictive value in breast cancer examination.
Assuntos
Neoplasias da Mama/patologia , Linfonodos/patologia , Idoso , Neoplasias da Mama/metabolismo , Feminino , Humanos , Metástase Linfática , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Esterases/metabolismo , Linfonodos/enzimologia , Idoso , Células Apresentadoras de Antígenos/enzimologia , Axila , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/secundário , Carcinoma Intraductal não Infiltrante/enzimologia , Carcinoma Intraductal não Infiltrante/secundário , Carcinoma Lobular/enzimologia , Carcinoma Lobular/secundário , Feminino , Fluoresceínas/metabolismo , Humanos , Linfonodos/patologia , Metástase Linfática , Ativação Linfocitária , Pessoa de Meia-Idade , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Linfócitos T/enzimologiaRESUMO
Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Células HeLa , Humanos , Invasividade Neoplásica , Esferoides CelularesRESUMO
OBJECTIVE: Apoptosis is being increasingly regarded as a key component in the development and progression of atherosclerosis. Since it has become apparent that the immune system plays a predominant role in mediating atherogenesis, there has been a growing recognition that the evaluation of lymphocyte apoptosis may contribute to understanding a persistent altered immune and inflammatory response. The aim of the present study was to evaluate the apoptotic effect of lysophosphatidylcholine (LPC) on peripheral blood lymphocytes (PBL) derived from unstable angina (UA) patients, as compared to healthy donors. METHODS: PBL isolated from 27 healthy donors and 25 age matched UA patients were examined. Early apoptotic events induced by LPC in resting and phytohemagglutinin (PHA)-activated lymphocytes were evaluated by several apoptotic assays. The levels of intracellular reactive oxygen species (ROS) and the expression of apoptotic regulated proteins (Bcl-2 and Bax) were measured. RESULTS: LPC was found to induce apoptosis in normal activated lymphocytes, in a dose- and time-dependent manner, in association with an increase in intracellular ROS. In UA patients, an exposure of PHA-activated PBL to LPC triggered neither an increase in ROS generation, nor in the apoptotic manifestations, and was associated with a significantly lower ratio of Bax/Bcl-2 expression. CONCLUSION: Our results indicate that PBL isolated from UA patients may be resistant to apoptosis induction by LPC, resulting from oxidative stress challenge and dysregulation of apoptosis-related protein expression.
Assuntos
Apoptose/efeitos dos fármacos , Doença da Artéria Coronariana/imunologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Adulto , Idoso , Angina Instável/imunologia , Angina Instável/metabolismo , Angina Instável/patologia , Anexina A5/metabolismo , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Tamanho Celular/efeitos dos fármacos , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Feminino , Humanos , Técnicas In Vitro , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Coloração e RotulagemRESUMO
The Michaelis-Menten constants (K(m) and V(max)) operated by linear programming, were employed for detection of breast cancer. The rate of enzymatic hydrolysis of fluorescein diacetate (FDA) in living peripheral blood mononuclear cells (PBMC), derived from healthy subjects and breast cancer (BC) patients, was assessed by measuring the fluorescence intensity (FI) in individual cells under incubation with either the mitogen phytohemagglutinin (PHA) or with tumor tissue, as compared to control. The suggested model diagnoses three conditions: (1) the subject is diseased, (2) the diagnosis is uncertain, and (3) the subject is not diseased. Out of 50 subjects tested, 44 were diagnosed correctly, in 5 cases the diagnosis was not certain, and 1 subject was diagnosed incorrectly.
Assuntos
Neoplasias da Mama/diagnóstico , Técnicas e Procedimentos Diagnósticos/estatística & dados numéricos , Programação Linear/estatística & dados numéricos , Feminino , Humanos , IsraelRESUMO
Estrogen-induced apoptosis has become a successful treatment for postmenopausal metastatic, estrogen receptor-positive breast cancer. Nitric oxide involvement in the response to this endocrine treatment and its influence upon estrogen receptor-positive breast cancer progression is still unclear. Nitric oxide impact on the MCF7 breast cancer line, before and after estrogen-induced apoptosis, was investigated in 3D culture systems using unique live-cell imaging methodologies. Spheroids were established from MCF7 cells vulnerable to estrogen-induced apoptosis, before and after exposure to estrogen. Spheroids derived from estrogen-treated cells exhibited extensive apoptosis levels with downregulation of estrogen receptor expression, low proliferation rate and reduced metabolic activity, unlike spheroids derived from non-treated cells. In addition to basic phenotypic differences, these two cell cluster types are diverse in their reactions to exogenous nitric oxide. A dual effect of nitric oxide was observed in the breast cancer phenotype sensitive to estrogen-induced apoptosis. Nitric oxide, at the nanomolar level, induced cell proliferation, high metabolic activity, downregulation of estrogen receptor and enhanced collective invasion, contributing to a more aggressive phenotype. Following hormone supplementation, breast cancer 3D clusters were rescued from estrogen-induced apoptosis by these low nitric oxide-donor concentrations, since nitric oxide attenuates cell death levels, upregulates survivin expression and increases metabolic activity. Higher nitric oxide concentrations (100nM) inhibited cell growth, metabolism and promoted apoptosis. These results suggest that nitric oxide, in nanomolar concentrations, may inhibit estrogen-induced apoptosis, playing a major role in hormonal therapy. Inhibiting nitric oxide activity may benefit breast cancer patients and ultimately reduce tumor recurrence.
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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.
Assuntos
Células Sanguíneas/citologia , Técnicas de Cultura de Células , Técnicas Analíticas Microfluídicas , Microfluídica , Humanos , Microfluídica/instrumentação , Células U937RESUMO
Reactive oxygen species (ROS) have recently been shown to be involved in multiple physiological responses through modulation of signaling pathways. Inappropriate production of these radicals, and their metabolites, leads to the development of various pathologies. Free radicals can induce both positive and negative effects in cells, and their metabolic pathways are very complex. Hence, it is crucial to be able to simultaneously and directly determine their production dynamics and concentrations in individual living cells, in physiological or pathological states, and in response to drugs. The aim of the present study was to monitor in real time the rates of ROS generation in promonocytic cells upon stimulation with hydrogen peroxide and oxidized lipid. Quantitative detection of intracellular ROS concentration in intact living U937 cells was performed by fluorescence intensity (FI) and polarization (FP) measurements utilizing the Optical LiveCell Array technology. The "dihydro" derivative probes of fluorescein (DCF-DA) and rhodamine (DHR123) were used to assess the intracellular levels of ROS. Each probe molecule exhibited a characteristic FI and FP in its non-fluorescent or oxidized form. Analysis of the temporal relationship between the kinetics of ROS generation and the onset of changes in mitochondrial membrane potential shows high variability within a cell population with regard to both processes. The data demonstrated that temporal measurement of ROS generation, in identifiable individual cells, reveals kinetic behavior that otherwise would be undetected.
Assuntos
Membranas Mitocondriais/fisiologia , Espécies Reativas de Oxigênio/análise , Análise Serial de Tecidos/métodos , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/sangue , Fluoresceínas/química , Humanos , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Potenciais da Membrana , Microscopia de Fluorescência , Microscopia de Polarização , Membranas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodamina 123/química , Células U937RESUMO
The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.
Assuntos
Algoritmos , Análise por Conglomerados , Biologia Computacional/métodos , Biologia Computacional/estatística & dados numéricos , Citometria de Fluxo , Citometria de Fluxo/estatística & dados numéricos , Polarização de Fluorescência/estatística & dados numéricos , Humanos , Células Jurkat , Ativação Linfocitária , Linfócitos T/imunologiaRESUMO
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 (K(m) and V(max)) 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 K(m) values after cell incubation with tumor tissue. It was also demonstrated that higher K(m) 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.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/imunologia , Leucócitos Mononucleares/enzimologia , Linfócitos/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Feminino , Humanos , Cinética , Pessoa de Meia-Idade , Mitógenos , Prognóstico , Medição de Risco , Células Tumorais CultivadasRESUMO
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.
Assuntos
Apoptose , Tamanho Celular , Linfócitos/fisiologia , Inibidores de Caspase , Humanos , Células Jurkat , Coloração e RotulagemRESUMO
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 death 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 radical 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.
Assuntos
Apoptose/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Lipoproteínas LDL/administração & dosagem , Relação Dose-Resposta a Droga , Humanos , Células Jurkat , Oxirredução , Células U937RESUMO
Living cells are known to exhibit great morphological, functional, spatial and temporal heterogeneity. Hence, the study of cells in a bulk, whether this bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results. An advantageous approach would rather be a comprehensive study of cell biological activity in single isolated living cells. In this study, we present an imaging approach for studying pre-apoptotic and very early apoptotic events, during cell death induced by lysophosphatidylcholine (LPC) at the single cell level. The aim of this study is to investigate intracellular events, such as the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) formation, before and immediately after LPC introduction to the lymphocytes at the level of individual cells. A new protocol of sequential staining was developed to study the relation between early apoptosis signs (PS externalization), MMP changes and intracellular ROS production rates at an individual Jurkat cell resolution. Simultaneous kinetic assessments of MMP, intracellular ROS levels and phosphatidylserine (PS) externalization were performed at a single cell resolution, using Optical LiveCell™ Array technology and image analysis. The parameters were measured and analyzed both before and during exposure to inducers in a Jurkat cell population, including three groups of single cells: spontaneous apoptotic cells, induced apoptotic cells and fully functional living cells. Exogenous LPC caused a heterogeneous intracellular response among Jurkat cells immediately after its introduction. Subgroups of cells with opposite changes of MMP and different kinetics of ROS increase, were revealed within the whole cell population. The subset of apoptosis-induced Jurkat cells, which became apoptotic within 3h after the LPC introduction, exhibited higher initial MMP compared to fully functional or spontaneous apoptotic cells. LPC-induced apoptosis was accompanied by a concomitant increase in intracellular ROS levels. In the present study, a method is described to assess the intracellular events in cells which were initially different in their physiological status. The individual T lymphocytes (Jurkat cells) in vitro have various susceptibilities to LPC effects at the very early stage of contact with the inducer. The apoptotic effect of LPC in individual Jurkat cells is associated with a relatively higher initial MMP before the introduction of the inducer and with a faster ROS formation within the affected cells. Such divergence may be significant in regulating the balance of lymphocyte subsets in pathological sites, either maintaining or preventing the inflammation components of atherosclerosis. We conclude that the presented approach provides the researcher, not only with the cell retaining methodology, but with opportunities to observe and find the distinctive cell subsets within the whole cell population as well, thus helping to define more exactly the role and importance of such sub-populations in physiological or pathological conditions.
Assuntos
Apoptose/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Células Jurkat , Cinética , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microscopia de Vídeo , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Rodaminas/química , Rodaminas/metabolismo , Fatores de TempoRESUMO
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.