The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Fluctuation of Information Entropy Measures in Cell Image

A simple, label-free cytometry technique is introduced. It is based on the analysis of the fluctuation of image Gray Level Information Entropy (GLIE) which is shown to reflect intracellular biophysical properties like generalized entropy. In this study, the analytical relations between cellular thermodynamic generalized entropy and diffusivity and GLIE fluctuation measures are explored for the first time. The standard deviation (SD) of GLIE is shown by experiments, simulation and theoretical analysis to be indifferent to microscope system “noise”. Then, the ability of GLIE fluctuation measures to reflect basic cellular entropy conditions of early death and malignancy is demonstrated in a cell model of human, healthy-donor lymphocytes, malignant Jurkat cells, as well as dead lymphocytes and Jurkat cells. Utilization of GLIE-based fluctuation measures seems to have the advantage of displaying biophysical characterization of the tested cells, like diffusivity and entropy, in a novel, unique, simple and illustrative way

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

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