Influence of matricial molecules on growth and differentiation of entrapped chondrocytes.

Primary cultures of rabbit articular chondrocytes have been cultivated normally and within three-dimensional systems using different alginate matrices. The in vitro proliferation capacity of the cells immobilized in the calcium alginate beads was investigated. The growth curve showed that chondrocytes are able to grow and to divide for several days inside the beads; in parallel an increase in protein contents was also measured. The differentiated phenotype of rabbit articular chondrocytes consists of cartilage-specific proteoglycans. During serial monolayer cultures this phenotype was lost and replaced by a low level of proteoglycan synthesis. On the contrary when cultivated in beads, entrapped cells maintained their differentiated phenotype over time; the rates of proteoglycan were similar to those of primary chondrocytes. All these parameters were tested comparatively using different substrata in monolayer cultures and in alginate gels. Assays were carried out to assess the influence of type I collagen, type IV collagen, and of fibronectine on the growth as well as on the differentiation phenotype. The encapsulation methodology is readily applicable to the culture of chondrocytes in single beads, in multiwell dishes, or to mass culture for a bioproduction of extracellular matrix components.

Implementation of biological elements in life support systems: rationale and development milestones.

To ensure the complete autonomy of man in space, biological life support technologies must be developed. A survey of today's knowledge in biotechnology has been performed and technologies suited to long-duration manned missions in space have been identified. Regardless of whether they are to be used in a space vehicle or at a lunar or planetary base, most of those technologies require development times of 20 to 30 years. It is therefore essential to start development now to ensure that the life support system is ready when development of Moon or Mars bases begins.

Immobilized articular chondrocytes: in vitro production of extracellular matrix compounds.

Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads. Both free and entrapped cells were allowed to grow under normal conditions. After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium. After long-term immobilization, the morphology and the viability of immobilized rabbit articular chondrocytes were preserved: cells remained viable and were able to grow and divide for several days inside the alginate beads in a culture incubator. The percentage of viable cells did not significantly decrease when immobilized cells were stored at 4 degrees C for 30 days. The basic metabolic properties (glucose consumption) and characteristic activities (proteoglycan secretion) were similar to those of free adherent cells with a time-dependent increase. A large scale bioproduction of extracellular matrix components may be considered of great interest for the ready-to-use complete culture systems of mammalian cells with high densities. Moreover immobilized forms also facilitate the use of cells in a bioreactor or in some unusual conditions (parabolic flights).

Effect of alginate and calcium on secretion of IL-1 by immobilized Swiss peritoneal macrophages.

Murine peritoneal macrophages were immobilized in calcium alginate gel macrobeads, using 1.5% Na-alginate and 50 mM CaCl2. Secretion of IL-1 by immobilized macrophages increased during time and reached 10 fold than IL-1 quantities secreted by adherent macrophages. Calcium and alginate individually enhance production of IL-1 by macrophages and act in synergy when macrophages are immobilized in calcium alginate matrix.

Alginate immobilized mammalian neurons: a potential tool to isolate new neuronal ligands.

We developed improved immobilization conditions which permitted (i) to immobilize neuroblastoma cells (N18) in calcium-alginate gel beads, (ii) to test the function of ionic channels using patch-clamp electrophysiological techniques and (iii) to quantitatively analyze ligand interactions with voltage-dependent sodium channels in neurons inside the beads. These results qualify this immobilization technique for the isolation and/or purification of ligands specific for neuronal cells.

Rabbit articular chondrocytes in alginate gel: characterisation of immobilized preparations and potential applications.

Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads. Both free and entrapped cells were allowed to grow under normal conditions. After long-term immobilization, the cells still exhibited metabolic activities, patterns of division, synthesis and secretion of extracellular matrix macromolecules such as type II collagen and proteoglycans. After 38 days, immobilized rabbit articular chondrocytes predominantly expressed type II but not type I collagen. Thus, they maintained their cartilage pheno-type. After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium. On the basis of these results, long-duration storage and large-scale production of extracellular matrix components are being investigated.

Storage and growth of neuroblastoma cells immobilized in calcium-alginate beads.

Mouse neuroblastoma cells (N18) were immobilized in calcium-alginate gel beads. Under standard culture conditions (37 degrees C; 5% CO2), cell growth was observed inside the beads. The number of cells increased threefold during 7 days of culture with cell division and differentiation visualized by electron microscopy. Cell properties maintained after short-term storage (2-3 days at 4 degrees C) included: (i) properties of voltage-dependent ionic channels tested by patch-clamp electrophysiological techniques; (ii) expression of cell-adhesion membrane proteins tested by immunohistochemistry (iii) morphological differentiation obtained by depletion of foetal calf serum in culture medium. The advantages of such an immobilization technique as applied to neurone cells are discussed.

gamma-Hexachlorocyclohexane inhibition of the calcium fluxes at the desensitized mouse neuromuscular junction.

The peripheral neurotoxicity of lindane was studied in vitro on mouse phrenic-diaphragm preparation. The experiments were performed on normal and denervated preparations. Twitches evoked by direct or indirect electrical stimulations and contractions evoked by external application of cholinergic agonists were compared in the presence or absence of lindane in the bath medium. Histochemical localizations of calcium were made on fibers after chemical stimulations. The results indicated that, at the normal endplate or at extrajunctionnal sites after denervation, there was a change in the intracellular Ca2+ distribution in response to external application of concentrated cholinergic agonists. This modification caused a transient contracture and could be visualized either by 45Ca autoradiography or by Alizarin Red S intracellular precipitations. Lindane inhibited the ACh-dependent contracture (I50 less than 10(-5) M) and suppressed the histochemical localizations of calcium. These lindane effects were observed on both normal and denervated muscles. It was concluded that lindane inhibited the Ca2+ influx presented by the desensitized ACh-sensitive areas of the muscle.

[In vitro demonstration of the effect of lindane on isolated synaptosome preparations]. / Mise en évidence in vitro d'unme action du lindane sur des préparations de synaptosomes isolés.

Lindane neurotoxicity is studied in vitro on Rat brain synaptosomes. Calcium uptake of isolated synaptosomes increases in the presence of pesticide; lindane action is observed on both normal and depolarized preparations.

Use of alizarin red S for histochemical staining of Ca2+ in the mouse; some parameters of the chemical reaction in vitro.

Alizarin red S (ARS) is used for histological characterization of calcium. In order to improve the conditions of the use of the dye on biological materials, the chemical reaction between ARS and Ca2+ is studied in vitro. In aqueous solution ARS and Ca2+ ions precipitate to form brick-red deposits. For precipitation to occur a neutral pH (4 less than pH less than 8) is required; the stoichiometric ratio is 1:1. ARS reacts with calcium via its sulfonate and hydroxyl groups. The apparent solubility product is around 10(-7) Na+ and K+ ions do not interfere with equilibrium of the precipitation reaction but Mg2+ and soluble proteins such as bovine serum albumin react with ARS and form soluble complexes which can lead to a decrease in the ARS quantity available to react with the calcium ions.

[Intracerebral distribution of lindane in rats: short term observation after administration of a single dose]. / Distribution intracérébrale du lindane chez le rat; observation à court terme après administration d'une dose unique.

Residual radioactivity in Rat brain structures is studied after single dose 14C Lindane administration. Short time after injection, at the onset of convulsions, white matter and myelinated structures show important retention, nucleated cells do not. This heterogeneous distribution agrees with the lipophilic behaviour of the pesticide.

[Distribution of C14-lindane in the rat after administration of a single dose by the intraperitoneal and intravenous route]. / Distribution du 14C-lindane chez le rat après administration d'une dose unique par voies intrapéritonéale et intraveineuse.

14C-Lindane retentions in Rat tissues were studied until 24 hrs after a single dose pesticide administration. Each organ shows particular kinetics. Adipose tissue is the most active in pesticide fixation but the lungs retain retain momentarily a large fraction of Lindane after intravenous injection.

Some actions of purified toxins from Bungarus fasciatus venom on isolated pig-heart mitochondria.

I. In the presence of succinate as an oxidation substrate, neurotoxins alpha, beta and gamma induce the following. Firstly, an increasing stimulation of oxygen uptake, which in potentiated by 25 muM Ca2+, Mg2+ 1.3 mM completely inhibits the effect of toxin alpha but not of toxins beta and gamma. Secondly, a depletion of the Mg2+, Ca2+ and K+ content of the water-soluble and membrane-bound mitochondrial compartments, following complex kinetics, which suggest a multistep interaction mechanism of the toxins with the mitochondria. 25 muM Ca2+ also potentiates the effect of the toxins on these ionic flows. Thirdly, no decrease of turbidity with toxin alpha, and a limited decrease with toxins beta and gamma. 2. In the absence of respiration, the neurotoxins induce a cationic depletion, the kinetics of which are different than with succinate, suggesting an instantaneous maximal effect on the inner membrane. Toxins beta and gamma (but not alpha) induce, under these conditions, a turbidity decrease of large amplitude, which is proportional to the amount of toxin added and tends to reach a maximum. With gamma toxin this turbidity decrease is faster than the rate of water uptake (which never exceeds 18%) indicating that it is due rather to structural modifications than to swelling. The same is observed with beta toxin, provided the mitochondrial protein concentration to be lower than 0.7 mg/ml. For higher concentrations, a continuous decrease of turbidity with a considerable uptake of water probably reflects the onset of phospholipasic activities. 3. It is postulated that structural modifications of the mitochondrial membranes are initiated which lead to the loss of their selective impermeability. The simultaneous loss of respiratory control with succinate may be due to the direct (though Ca2+-potentiated) displacement of the fraction of the membrane-bound Mg2+ ions which controls its energy-transducing properties. 4. In addition, correlations between the effects of the toxins on mitochondria and their neurotoxicity in vivo are discussed.