Quantification of Etoricoxib in Low Plasma Volume by UPLC-PDA and Application to Preclinical Pharmacokinetic Study.

An ultra-performance liquid chromatography with photodiode array (UPLC-PDA) UV detection method was developed here for the first time for simple, rapid, selective and sensitive quantification of the commonly prescribed selective cyclooxygenase-2 (COX-2) inhibitor etoricoxib in low plasma volumes (50 µL). The method includes protein precipitation followed by liquid-liquid extraction, evaporation and reconstitution. A gradient mobile phase of 75:25 going to 55:45 (v/v) water:acetonitrile (1 mL/min flow rate) was applied. Total run time was 8 min, representing a significant improvement relative to previous reports. Excellent linearity (r2 = 1) was obtained over a wide (0.1-12 µg/mL) etoricoxib concentration range. Short retention times for etoricoxib (4.9 min) and the internal standard trazodone (6.4 min), as well as high stability, recovery, accuracy, precision and reproducibility, and low etoricoxib LOD (20 ng/mL) and LOQ (100 ng/mL), were achieved. Finally, the method was successfully applied to a pharmacokinetic study (single 20 mg/kg orally administered etoricoxib mini-capsule) in rats. In conclusion, the advantages demonstrated in this work make this analytical method both time- and cost-efficient for drug monitoring in pre-clinical/clinical settings.

Measurement of Water Saturation in Soybean Oil.

In a previous study, it was observed that survivability was low when attempting to cryopreserve sperm cells in a nanoliter-sized droplet protected under soybean oil, in stark contrast to the high survival rates in milliliter-sized droplets. In this study, infrared spectroscopy was used to provide an estimate of the saturation concentration of water in soybean oil. By following the time evolution of the infrared absorption spectrum of water-oil mixtures, the saturation of water in soybean oil was found to reach equilibrium after 1 h. From the absorption spectra of neat water and neat soybean oil and the application of the Beer-Lambert law to an estimation of the absorption of a mixture from its individual components, it was estimated that the saturation concentration of water is 0.010 M. This estimate was supported by molecular modeling using the latest semiempirical methods (in particular, GFN2-xTB). While for most applications the very low solubility has little impact, the implications in those exceptions were discussed.

Fluorophore spectroscopy in aqueous glycerol solution: the interactions of glycerol with the fluorophore.

A common perception exists that glycerol provides an inert-like environment modifying viscosity and index of refraction by its various concentrations in aqueous solution. Said perception is herein challenged by investigating the effects of the glycerol environment on the spectroscopic properties of fluorescein, as a representative fluorophore, using steady-state and time-resolved techniques and computational chemistry. Results strongly suggest that the fluorescence quantum yield, measured fluorescence lifetime (FLT), natural lifetime and calculated fluorescence lifetime are all highly sensitive to the presence of glycerol. Glycerol was found to impact both the ground and first excited states of fluorescein, quenching and modifying both absorption and emission spectra, affecting the fundamental electrical dipoles of the ground and first excited singlet states, and lowering FLT and quantum yield. Furthermore, the Stern-Volmer, Lippert-Mataga, Perrin and Strickler-Berg relations indicate that glycerol acts upon fluorescein in aqueous solution as a quencher and alters the fluorescein geometry. Predictions made by computational chemistry impressively correspond to experimental results, both indicating changes in the properties of fluorescein at around 35% v/v aqueous glycerol, a clear indication that glycerol is not an innocent medium. This study proposes the Strickler-Berg relation as a means of detecting non-negligible effects of a hosting medium on its host fluorophore. These new insights on the molecular structures, the interactions between glycerol and its host fluorophore, and the effects of one on the other may be essential for understanding fundamental phenomena in chemistry and related fields.

Co-culture hydrogel micro-chamber array-based plate for anti-tumor drug development at single-element resolution.

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.

Discrimination of leukemic Jurkat cells from normal lymphocytes via novo label-free cytometry based on fluctuation of image gray values.

We introduce a simple, label-free cytometry technique, based on the spatio-temporal fluctuation analysis of pixel gray levels of a cell image utilizing the Gray Level Information Entropy (GLIE) function. In this study, the difference in GLIE random fluctuations and its biophysical etiology in a comparison cell model of leukemic Jurkat cells and human healthy donor lymphocytes was explored. A combination of common bright field microscopy and a unique imaging dish wherein cells are individually held untethered in a picoliter volume matrix of optical chambers was used. Random GLIE fluctuations were found to be greater in malignant Jurkat cells than in benign lymphocytes, while these fluctuations correlate with intracellular vesicle Mean Square Displacement (MSD) values and are inhibited by myosin-2 and adenosine triphosphate (ATP) inhibitors. These results suggest that the incoherent active forces acting on the cytoskeleton which cause mechanical dissipative fluctuation of the cytoskeletal and related intracellular content are the biophysical cellular mechanism behind the GLIE random fluctuation results. Analysis of the results in Jurkat cells and normal lymphocytes suggests the possible potential of this simple and automated label-free cytometry to identify malignancy, particularly in a diagnostic setup of multiple cell examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates.

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.

The cationic dye basic orange 21 (BO21) as a potential fluorescent sensor.

The present study investigates the fluorescence properties of BO21 and their dependence on various intracellular conditions. The results obtained with cell-free solutions indicate that the influences of pH and temperature on the fluorescence spectra are negligible, while viscosity, various proteins and heparin have significant influence. In the presence of heparin, a red shift of the emission spectrum (from 515 to 550 nm) is observed, suggesting that this shift cannot simply be attributed to electrostatic interaction between BO21 and the polyanionic heparin, but rather to aggregation of BO21 on the polyanion. In water, the quantum yield of BO21 was found to be 1000 times lower than that of fluorescein, yet surprisingly its fluorescence polarization (FP) was found to be about 40 times higher (FP = 0.470), even though both have similar structures and molecular weights. A thorough analytical and experimental investigation of these phenomena indicates that the very high FP of BO21 in water is a consequence of its very short lifetime. However, upon the addition of heparin to aqueous BO21, the fluorescence lifetime (FLT) of BO21 increases from τ = 10.35 to 56.5 ps, with a consequent dramatic drop in its fluorescence polarization from 0.470 to 0.230. From its behavior in aqueous glycerol solution, it is hypothesized, with support from theoretical calculations, that BO21 is a molecular rotor. Using these properties, BO21 may be a good candidate as a sensor, for example, of heparin levels in blood or of intracellular viscosity.

Nitric oxide is cytoprotective to breast cancer spheroids vulnerable to estrogen-induced apoptosis.

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.

In situ label-free static cytometry by monitoring spatiotemporal fluctuations of image gray values.

Spatiotemporal fluctuation of homogeneity and randomness of gray values within an image was explored and utilized as a label-free means for cell examination. This was done by utilizing a user-friendly combination of simple bright field microscope and Cytocapture dish, wherein cells are individually held, each within a picoliter optical chamber, forming an array of cells to be repeatedly measured over time and biomanipulated in situ at single-cell resolution. First, the measured gray level information entropy (GLIE) was used and, based on the fact that living cells are not in a state of thermodynamic equilibrium but rather in a metastable state, two fluctuation-sensitive measures were proposed and examined: ASDE­the spatial average of temporal standard deviation (SD) of GLIE, and AA­the average time autocorrelation of GLIE. System performance was validated on cell-free solutions. This was followed by examining the performance of the measures AGLIE, ASDE, and AA to distinguish among individual live-still, dead and live cells from various cell lines, as well as between cells which were and were not induced to differentiate. Results, which were obtained on four types of cells, indicate advantages of the proposed measures which are believed to be significant additions to the microscope-based probe-free toolbox.

Analysis of the Spectroscopic Aspects of Cationic Dye Basic Orange 21.

Spectroscopic properties of cationic dye basic orange 21 (BO21) in solutions, in solids, and within leukocytes were examined. Results obtained with solutions indicate that influence of variables such as pH, viscosity, salt composition, and various proteins on the absorption spectrum of BO21 is negligible. However, in the presence of heparin, a blue shift (484-465 nm) is observed, which is attributed to the aggregation of BO21 on the polyanion. Applying density functional theory demonstrates that in aqueous solutions (a) the formation of BO21 oligomers is thermodynamically favorable, they are oriented in an antiparallel dipolar arrangement, and their binding energies are lower than those of parallel dipolar arrangements, (b) association between BO21 aggregates and heparin is highly favorable, and (c) the blue shift is due to the mixing of π → π* transitions caused by BO21 molecule stacking. However, when embedded in basophils, the absorption spectra of intracellular BO21 is extremely red-shifted, with two peaks (at 505 and 550 nm) found to be attributed to BO21 and the BO21-heparin complex, respectively, which are intracellularly hosted in nonaqueous environments. Initial evidence of the ability to differentiate between leukocyte types by BO21 is presented.

Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array.

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.

Photobleaching of fluorescein as a probe for oxidative stress in single cells.

BACKGROUND: ROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis. METHODS: Photobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H2O2). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant. RESULTS: Fluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H2O2 at 50 µM. At higher H2O2 concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H2O2 in phosphate buffer saline (PBS), where the addition of H2O2 decreases the decay time, regardless of the irradiation dose used. CONCLUSIONS: The anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods. SIGNIFICANCE: Assessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.

Real-time monitoring of changes in plasma membrane potential via imaging of fluorescence resonance energy transfer at individual cell resolution in suspension.

A method for monitoring heterogeneity in changes of plasma membrane potential (PMP) at an individual cell resolution while in suspension, utilizing a simple and low-cost wide-field illumination arrangement, is presented. The method is modeled via HEK-293 cell line in suspension, double stained with coumarin and oxonol (donor and acceptor), which were loaded into an array of nanoliter wells, each designed to preserve the individuality of the nontethered cell it holds during vigorous biomanipulation. Depolarization of PMP was induced by high K(+) solution, reducing the proximity between the membrane fluorophores and subsequently reducing the efficiency (E%) of resonance energy transfer between them. Spatial plots of E% were produced from both images of fluorescence intensity and polarization. The spatial resolution of E% plots seem to be higher, and their contrast greater, when calculated from the polarization, rather than from the intensity of the fluorescence.

Nanoparticulate flurbiprofen reduces amyloid-ß42 generation in an in vitro blood-brain barrier model.

INTRODUCTION: The amyloid-ß42 (Aß42) 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. METHODS: The Aß42 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. RESULTS: PLA-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 Aß42 levels in the abluminal compartment of the BBB model. CONCLUSIONS: In 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 Aß42 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.

The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine.

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.

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.

Whole-object fluorescence lifetime setup for efficient non-imaging quantitative intracellular fluorophore measurements.

In the present study we introduce a Whole-Object Fluorescence Life Time (wo-FLT) measurement approach for ease and a relatively inexpensive method of tracing alterations in intracellular fluorophore distribution and in the physical-chemical features of the microenvironments hosting the fluorophore. Two common fluorophores, Rhodamine 123 and Acridine Orange, were used to stain U937 cells which were incubated, with and without either Carbonyl cyanide 3-chlorphenylhydrazon or the apoptosis inducer H(2)O(2). The wo-FLT, which is a non-imaging quantitative measurement, was able to detect several fluorescence decay components and corresponding weights in a single cell resolution. Following cell treatment, both decay time and weight were altered. Results suggest that the prominent factor responsible for these alterations and in some cases to a shift in emission spectrum as well, is the intracellular fluorophore local concentration. In this study it was demonstrated that the proposed wo-FLT method is superior to color fluorescence based imaging in cases where the emission spectrum of a fluorophore remains unchanged during the investigated process. The proposed wo-FLT approach may be of particular importance when direct imaging is impossible.

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

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.

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.