Xavier Ronot

Cytometric assessment of mitochondria using fluorescent probes

Mitochondria are most important organelles in the survival of eukaryotic aerobic cells because they are the primary producers of ATP, regulators of ion homeostasis or redox state, and producers of free radicals. The key role of mitochondria in the generation of primordial ATP for the survival and proliferation of eukaryotic cells has been proven by extensive biochemical studies. In this context, it is crucial to understand the complexity of the mitochondrial compartment and its functionality and to develop experimental tools allowing the assessment of its nature and its function and metabolism. This review covers the role of the mitochondria in the cell, focusing on its structure, the mechanism of the mitochondrial respiratory chain, the maintenance of the transmembrane potential and the production of reactive oxygen species. The main probes used for mitochondrial compartment monitoring are described. In addition, various applications using mitochondrial‐specific probes are detailed to illustrate the potential of flow and image cytometry in the study of the mitochondrial compartment. This review contains a panel of tools to explore mitochondria and to help researchers design experiments, determine the approach to be employed, and interpret their results.

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αvβ3 integrin localization

SummaryThis study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the αvβ3 integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the αvβ3 integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the αvβ3 integrin distribution.

Chalcones Derivatives Acting as Cell Cycle Blockers: Potential Anti Cancer Drugs?

Chalcones (1,3-diphenylpropen-1-ones) are naturally occurring compounds belonging to the flavonoid family and are largely investigated in various therapeutic area and especially as antitumor drugs. In the latter field, the literature survey indicates that effect on the cell cycle is one of the most important targets domains of chalcones. In this review, we will shed light on: a) the structural criteria responsible for the cell cycle perturbations, b) the activity of chalcones on cell cycle molecular players or regulators c) the correlation between the chalcone-structure and proteins involved directly or not in cell cycle regulation and apoptosis by enhancement of proapoptotic molecules expression. We will discuss some perspectives related on how can we deal with chemical modification of chalcones to come up with more potent compounds to provide new ways for cancer treatment.

Flow Cytometric Sorting of Living, Highly Motile Human Spermatozoa Based on Evaluation of Their Mitochondrial Activity

We investigated the applicability of flow cytometric (FCM) sorting to select, with no deleterious effects, fractions of living, highly motile spermatozoa after staining with rhodamine 123 (Rh123) and propidium iodide (PI) for assessment of their mitochondrial activity and viability, respectively. Sperm cells were subjected to FCM sorting according to their Rh123 fluorescence intensity, and computer-aided sperm analysis (CASA) for percentage motility and movement characteristic measurements was carried out on the entire sperm populations and on the Rh123-positive (Rh123+) sorted fractions. A first experiment on five sperm samples from fertile donors pre-selected by either swim-up or simplified Percoll gradient indicated no detrimental effect of the FCM sorting procedure because: (a) the numbers of Rh123+ motile sperm were not decreased by FCM sorting; (b) data on the sorted fractions showed a tendency (not significant) for an increase in movement parameters rather than a drop; and (c) a significant decrease in the percentage of PI-positive (PI+) sperm (13% vs 3%; p < 0.05) was measured. A second experiment was performed on sperm samples from four patients, only washed and re-suspended in B2 medium. This demonstrated a significant increase in some characteristics of movement quality related to a substantial and selective immobilization of the less motile sperm. The significant drop in the percentage of PI+ sperm after FCM sorting (p < 0.01) was less pronounced than after FCM sorting of pre-selected sperm (12% vs 3%, respectively), indicating a lethal effect of FCM sorting on a small proportion of presumably moribund sperm. These preliminary data indicate a differential effect of FCM sorting on sperm according to their function characteristics and suggest the potential importance of these methods for the characterization in vitro of sperm subpopulations on the basis of functional criteria.

Investigation of Chalcones as Selective Inhibitors of the Breast Cancer Resistance Protein: Critical Role of Methoxylation in both Inhibition Potency and Cytotoxicity

ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC(50) values below 0.5 μM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6-hydroxyl-2,4-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.

Characterization of cell deformation and migration using a parametric estimation of image motion

This paper deals with the spatio-temporal analysis of two-dimensional deformation and motion of cells from time series of digitized video images. A parametric motion approach based on an affine model has been proposed for the quantitative characterization of cellular movements in different experimental areas of cellular biology including spontaneous cell deformation, cell mitosis, individual cell migration and collective migration of cell populations as cell monolayer. The accuracy and robustness of the affine model parameter estimation, which is based on a multiresolution algorithm, has been established from synthesized image sequences. A major interest of the authors approach is to follow with time the evolution of a few number of parameters characteristic of cellular motion and deformation. From the time-varying eigenvalues of the affine model square matrix, a precise quantification of the cell pseudopodial activity, as well as of cell division has been performed. For migrating cells, the motion quantification confirms that cell body deformation has a leading role in controlling nucleus displacement, the nucleus itself undergoing a larger rotational motion. At the cell population level, image motion analysis of in vitro wound healing experiments quantifies the heterogeneous cell populations dynamics.

Effects of donor's age on growth kinetics of rabbit articular chondrocytes in culture

The in vitro proliferative capacity of articular chondrocytes derived from young and old rabbits was investigated to examine if the modifications incurred can be related to the in vivo aging. Determinations were made of the cartilage cell density, cell volume, cell number at confluency, plating efficiency, growth curve and DNA content distributions. The old donor cells were characterized by a decline in all the parameters of cartilage growth studied: cell number at confluency, cell replication rate (from 20 h to 45 h) as well as an increase in cell volume. The mean cycle time in vitro increased from 17.5 h compared to 27 h during in vivo aging, essentially because of an elongation of the G1 phase. Chondrocytes derived from young and old donors may be an appropriate model system for studying the in vitro effects of drugs on rheumatoid diseases as a function of in vivo aging.

Age-related changes in rabbit articular chondrocytes

Cell culture techniques have been used extensively in the study of the aging process at the cellular level. The senescent articular chondrocyte seems to be a good model to examine the responses to aging in osteoarthritis, one of the most frequent diseases of old age. Thus in vitro chondrocyte senescence, established by weekly subculture was characterized by a declining proliferation rate during late passages, from a rapid growth rate in early subculture to a complete loss of the proliferation capacity after 8 +/- 1 passages. Flow cytometric analysis show a time course decrease in the fraction S and G2 + M during the subculture, and a concomitant enhancement in protein content related to the increase of cell size. The immunocytochemistry assays revealed an appearance of a rigid cytoarchitecture with an increase in the number, and organization, of three cytoskeletal components: actin, tubulin and vimentin. The cultured chondrocytes therefore undergo in vitro aging analogous to that described for diploid fibroblasts, and could constitute a cellular model for pharmacological and toxicological assays.

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.