Monique Adolphe

A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.

A simple and sensitive cell-cell adhesion microplate assay was established using the cytoplasmic fluorescent dye, calcein AM. The procedure involves three steps: the labeling of lymphocytes with an adequate concentration of calcein AM (20 microM) during a short incubation period (30 min); the adhesion of 2 x 10(5) labeled lymphocytes per well to confluent keratinocyte or fibroblast monolayers grown in microtiter plates for 90 min; and, finally, measurement of the fluorescent signal utilizing a new system of cold-light microfluorimetry (Rat, 1993). During the adhesion assay, the release of calcein from labeled lymphocytes is low and the method permits the detection of as few as 1000 adherent cells. This non-radioactive procedure takes less than 4 h to perform and has proven to be as accurate and reliable as the common method using radioactive isotopes. In addition to its simplicity, the use of a fluorescent molecular probe in conjunction with cold-light microfluorimetry (CLF) offers many advantages of safety and economy, and can readily be adapted to the different cell types that participate in cell-cell adhesion.

Dedifferentiated chondrocytes cultured in alginate beads: Restoration of the differentiated phenotype and of the metabolic responses to Interleukin‐1β

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin‐1β, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92‐kDa gelatinase in response to interleukin‐1β was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin‐1β on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin‐1β and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis. J. Cell. Physiol. 176:303–313, 1998.

Culture and characterization of juvenile rabbit tenocytes

The culture of rabbit tenocytes could be a useful model in the study of the physiopathology and pharmacotoxicology of tendons. This work was undertaken to examine the in vitro behavior of tenocytes from juvenile rabbit Achilles tendons. We report observations of the morphological and biological characteristics of primary culture and subsequent passages of rabbit tendon cells cultured in monolayer. Data obtained by electron microscopy and growth curves were complementary. After 36 passages, the generation time of tenocytes did not change and no sign of senescene could be seen. Primary culture and the first passages retained the expression of tenocyte differentiated functions, synthesis of type I collagen and decorin. Cell growth behavior was not modified upon passaging. However, when subcultured, tenocytes displayed a modulated phenotype.

New in vitro fluorimetric microtitration assays for toxicological screening of drugs.

Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR>900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl4). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh 123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.

In vitro study of cytotoxicity of quinolones on rabbit tenocytes

Tendinitis and tendon rupture complicating fluoroquinolone therapy have been reported recently, especially affecting men over 60 years. These new quinolones are more potent antimicrobial agents than older nonfluorinated compounds like nalidixic acid. We compared the effects of one quinolone (nalidixic acid) and two fluoroquinolones (norfloxacin and pefloxacin) on cultured rabbit Achilles tendon cells. First, we examined their effects on cell viability, mitochondrial succinate dehydrogenase and global activity, mitochondrial activity using microtitration methods. Pefloxacin and norfloxacin were more cytotoxic than nalidixic acid according to IC50 values. These results confirm that mitochondria represent a biological target of fluoroquinolones. Moreover, the extracellular matrix was studied by molecular hybridization. After a 72 h treatment, the level of type I collagen transcripts was not modified with any of the three antimicrobial agents, whereas mRNA encoding decorin was decreased with 10-4 mol/L pefloxacin only. The decrease of transcripts encoding decorin suggests that this matrix component is another target of pefloxacin and modification of decorin seems to be an early event (before mitochondrion alteration) which may contribute to the explanation of tendon rupture.

Induction of DNA synthesis in rat macrophages in vitro by inflammatory exudate

THE proliferation of macrophages in inflammatory exudates1–3 contrasts with the failure of these cells to divide in vitro except in the presence of highly specialised conditioned media4–7. Macrophage division in vivo, however, could be explained if inflammation produced a local mitogenic factor.

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.

Growth-promoting effects of acidic and basic fibroblast growth factor on rabbit articular chondrocytes aging in culture

Rabbit articular chondrocytes have a limited growth potential in vitro. After four passages in culture, chondrocytes have accomplished more than 50% of their life span. At this stage of culture, they are considered to be senescent-like, since a dramatic decrease in proliferative capacity and enhanced cell size and protein content are observed. These aged cells are, however, still able to respond to fibroblast growth factor (FGF). The addition of either acidic or basic FGF (10 ng/ml) to culture medium permitted an enhanced proliferation. The attenuation of FGF mitogenic activity during aging was not observed for both fractions. Moreover, when treated with acidic or basic FGF, aged chondrocytes had a smaller size and a lower protein content. The acidic FGF was less potent than the basic FGF in delaying the evolution of aged chondrocytes to senescence.

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.

Type II transglutaminase expression in rabbit articular chondrocytes in culture: relation with cell differentiation, cell growth, cell adhesion and cell apoptosis

Depending on the cell type studied, the involvement of type II transglutaminase (TGase) has been proposed in almost any event of the cell life such as differentiation, apoptosis, growth, aging, cell morphology and adhesion, metastatic capacity or extracellular matrix stabilization. In order to define the field(s) where this enzyme may be implicated in chondrocytes, type II TGase expression was studied in chondrocytes at different passages which differentiated state was modulated by retinoic acid, dihydrocytochalasin B or staurosporin. Results showed that (i) type II TGase expression is not incompatible with type II collagen expression, a main marker of chondrocyte differentiation (ii) type II TGase expression is higher when cells are in the exponential phase of growth than when growth arrested (iii) a high type II TGase expression does not imply that cells are apoptotic although cell apoptosis correlates with increased type II TGase expression (iv) non-adherent cells do not express type II TGase whereas adherent cells do whatever their differentiation state as assessed by type II collagen synthesis. These results suggest that, in articular chondrocytes, type II TGase is specifically implicated in the cell adhesion capacity.