FR2735146A1 - Culturing marine invertebrate cells - Google Patents

Abstract

Culturing marine invertebrate cells comprises incubating isolated, viable, contaminant-free cells in a medium based on sterile sea water. The incubation temp., the compsn. and the physicochemical properties of the medium is selected so as to obtain viable cells and to maintain or improve their functional capacities for \-30 days. Also claimed are: (a) a process for the freeze/thaw treatment of a biological material comprising isolated cells as above, cells recovered after culture by the above process and the tissues and organs from which they are derived, which comprises contacting the biological material with a cryoprotectant, slowly cooling the material to a temp. below -20 deg C at a rate of 1-5 deg C/min. and immersing the material in liq. N2; (b) cell cultures and biological material obtd. by the above processes, and (c) kits for carrying out bioassays which contain a biological material or a cell culture as in (b) with an appropriate culture media and reagents.

Description

PROCESS FOR CULTURING INVERTEBRATE CELLS
MARINEES AND CULTURES OBTAINED.

 The invention relates to the establishment of cultures of marine invertebrate cells.

 It relates more particularly to a process for obtaining isolated cells of marine invertebrates and culturing these cells. It also targets the crops thus obtained, and their applications, particularly in biotests.

 Cell cultures, primary cultures and lineages, are experimental models with multiple applications in both basic and applied research.

 The first work in the field of marine invertebrate cell culture dates back nearly fifty years and focused on the cultivation, by explant, of oyster mantle.

 If Echinoderms (starfish and sea urchin), arthropods (especially shrimp), and some other marine invertebrates were used for experiments, it is among the molluscs and especially the bivalves that the animals were mainly chosen for the trials. culture.

 In vitro maintenance of isolated marine Invertebrate cells has been reported, more or less long term (from a few hours to several weeks), as well as bioassays allowing some short-term studies. But the value and the limits of the models put in place until now can not be evaluated rigorously considering the rare tests of functional capacity implemented during the duration of the cultures.

 By the choice of a set of parameters and conditions, the inventors have developed a protocol for isolating and culturing marine invertebrate cells, making it possible to perform these cultures routinely with in vitro maintenance of at least some of their biosynthesis activities.

 The object of the invention is therefore to provide a method for culturing marine invertebrate cells that is functional in the short term, but also in the longer term, without any problem of microbial contamination.

 It also aims to provide a method for freezing-thawing isolated cells to obtain cultures whose biosynthesis activity is preserved, this method being applicable, in general, the biological material used according to the invention.

 The invention also aims to provide cultures of these marine invertebrate cells and their uses, in particular as cell models in bioassays.

 The method for culturing marine invertebrate cells of the invention is characterized in that it comprises the incubation of isolated, viable, substantially contaminant-free cells at a temperature and in a medium whose composition, based on sterile seawater, and the physicochemical properties, in particular osmolarity and pH, are chosen so as to obtain viable cells and maintain or improve their functional capacities for at least 30 days.

 According to the invention, the control of the viability of the cells, carried out both at the level of the isolated cells used and the cells of the culture, is assessed by the Trypan blue test, as described in the examples, the quality cells being controlled by electron microscopy. To carry out this test, the osmolarity of Trypan blue was adjusted to that of seawater, of the order of 1100 mOsm / ml, by addition of a low molecular weight carbohydrate, in order to avoid formation. precipitates and a solution too viscous. As stated in the examples, albinose is particularly preferred.

 By "practically examples of contaminants" is meant cells freed from bacteria, protozoa, fungi, including yeasts, which most commonly contaminate marine Invertebrates.

 The functional capacities of the cells, whose study is carried out in order to follow and control the evolution of the cultures, are appreciated with regard to the biosynthesis of proteins, lipids and DNA, by visualization and measurement of the incorporation radiolabeled precursors, according to the protocols given in the examples.

 The isolated cultured cells are as obtained from explants or tissue dissociation and after a decontamination pre-treatment.

 The tissue fragments or explants are immersed in a suitable culture medium, the isolation of the cells being obtained by migration from the expirants.

 For dissociation of tissues, a chemical system and / or an enzymatic system and / or a mechanical system is used.

 The parameters involved in this step are related to each other and will be easily adjusted by those skilled in the art to obtain the desired results. Thus, when the concentration of dissociation agent is low, the treatment time must be lengthened. As regards the temperature, it is advantageous, more generally, to operate at low temperature, in particular at a temperature not exceeding 10 C, and more preferably of the order of 4 "C.

 A particularly suitable dissociation agent is pronase. The use of low concentrations of pronase, of the order of 0.020 to 0.030E, more especially 0.025E, for about 12 hours, at a temperature not exceeding about 40C makes it possible to obtain a satisfactory dissociation of the cells with a high viability rate, as confirmed by the checks carried out.

 Pre-treatment of tissues, and even animals, ensures decontamination of the materials used.

According to an advantageous arrangement of the invention, the decontamination of the tissues is carried out by treatment with the aid of antibiotics and, where appropriate, mucolytic agent.
Suitable antibiotics include penicillin, streptomycin, kanamycin, gentamycin, alone or in admixture.

 As a mucolytic agent, dithiothreitol is especially mentioned.

 The pre-treatment of the animals used to obtain the cells makes it possible to improve the decontamination. It is thus advantageously used brushing and / or the action of disinfectants such as alcohol 90 "and / or handling in a sterile atmosphere and / or washing with large volumes of sterile seawater and / or dissecting tissues under sterile conditions and / or treating the tissues with antibiotic and / or mucolytic baths.

 Advantageously, the animals collected are housed in hatchery, in seawater, for example for a fortnight, then left to rest in tanks fed with recycled seawater and sterilized by UV.

 To carry out the incubation step of the culture method of the invention, the cells are inoculated, the viability of which has been verified, for example using the Trypan blue test as mentioned above, because of the from about 1 to 2 million viable cells per ml of medium.

 As shown in the examples, a seeding rate of the order of 1.5 million / ml gives satisfactory results.

 The physicochemical parameters of the culture medium are chosen taking into account the viability tests and functional capacity studies carried out as a control. Thus, the osmolarity is of the order of 800 to 1300 mOsm, given the osmolarity of seawater and haemolymph, and advantageously of the order of 1100 mOsm. The pH of the medium is advantageously chosen according to the pH of the internal medium and that of the external medium. Values are generally in the range of 7.3 to 8 or higher. A buffer medium is preferably added to the culture media. By way of example, mention may be made of an organic buffer, such as the Hepes buffer.

 The seawater-based culture medium is advantageously supplemented with additives as present in the Leibovitz medium.

 This results in a favorable effect on cell adhesion and the maintenance of morphological characters.

 It is especially advantageous to supplement the seawater with Leibovitz-15 medium at a rate of about 10 to 20% (V / V), and in particular about 15% (V / V).

 According to another advantageous arrangement, the sea water is further supplemented with serum, in particular serum of mammalian origin. Concentrations which prove to be suitable are of the order of 5 to 15E (V / V), in particular of approximately 10% (v / v). Decomplemented serum is preferably used.

 According to yet another advantageous arrangement of the invention, the culture medium comprises lipids, more especially unsaturated lipids originally derived from microalgae, or at least one complementation factor of mammalian origin chosen from among hemolymphs, embryonic extracts. growth factors, such as EGF or bFGF, brain ganglion extracts.

 An incubation temperature generally suitable for carrying out the cultures is of the order of 15 to 16 ° C. However, other temperatures may be chosen depending on the results of the viability and cell biosynthesis capacity tests. cell types to grow.

 During the incubation, it operates under a normal atmosphere, not enriched in CO2.

 According to conventional techniques, the culture is carried out on a support, the supports made of plastic material proving satisfactory in this respect. Attachment factors can be used to promote cell adhesion.

 The environment is renewed during the culture.

 The various steps mentioned above are particularly suitable for the culture of embryonic cells or differentiated cells of hemocytes and cells of various organs such as, depending on the species, the mantle, the labial palps, the muscles, the gills, the digestive gland and the heart, which is a particularly preferred organ for carrying out the culture method of the invention.

This method is of general application, and will advantageously be used for the culture of cells of molluscs, echinoderms, arthropods and the like.
Marine invertebrates.

Examples of molluscs include bivalves, such as oysterids, mytilids and pectinids, gastropods and cephalopods; from
Echinoderms, Asteridae, Echinids, among the
Arthropods, crustaceans like shrimps.

 The invention also relates to a process for obtaining in frozen form a biological material as defined above and comprising the isolated cells, before seeding, the cells collected after a few days of culture, as well as the raw material that can be used in the context of producing cultures according to the invention, such as embryos and gametes.

 The biological material, in a culture medium as defined above, is brought into contact with a cryoprotective agent and is then frozen at a temperature of less than -200 ° C., advantageously less than -50 ° C., preferably of the order at -80 ° C., at a slow speed of between 1 and 5 ° C./min, and immersed in liquid nitrogen.

 A particularly preferred cryoprotectant is dimethylsulfoxide (DMSO).

 The contact time before freezing is especially 10 to 20 minutes.

 Thawing of the biological material is carried out by bringing the containers containing them at a temperature advantageously greater than ambient, preferably of the order of 30 to 400C, in particular about 370C.

 To carry out this step, the containers are immersed, for example, in a water bath at the chosen temperature.

 The freezing medium is then advantageously diluted with a nutrient culture medium, and the cells separated.

 For example, centrifugation is performed and, after recovery of the cell pellet, the cells are resuspended for seeding.

 As illustrated by the examples, this method makes it possible to obtain cells whose viability and biosynthetic activity are maintained relative to the cells obtained after the isolation step.

 The invention thus provides a basic material which facilitates the realization of cell cultures as indicated above and allows standardization of the tests carried out with these cultures.

 The cell cultures as obtained according to the method described above and the cells collected from these cultures as well as in general the biological material as treated according to the invention, in particular the biological material in frozen form also enter into the scope of the invention.

 The cell cultures are characterized in that they consist essentially of viable cells capable of neosynthesizing DNA and other products with a high added value, such as proteins and lipids, in particular the unsaturated lipids defined in the examples and put into practice. evident in the functional capacity tests described in these examples.

 The invention also aims, as new products, these proteins and lipids as identified by the tests described in the examples.

 The resulting crops can be cocultures. The invention aims in particular co-cultures of epithelial type cells and myocytes of Bivalves, including scallops.

 Feasibility studies concerning the use of cell cultures, cells and, in general, biological material as models in in vitro bioassays have demonstrated their interest in many applications.

 The cell cultures and cells of the invention, and generally said biological material, especially embryos and gametes in frozen form, are thus particularly advantageous as cellular models in vitro in toxicology and pathology.

 These models allow the realization of acute toxicity test on marine invertebrate cells, as obtained according to the techniques described above, whether fresh cells, frozen or in kit form.

 They are biomarkers of cytotoxic and genotoxic effects of xenobiotics. Examples include their application to the monitoring of seawater quality, sediments, the study of the effects of products on the environment, whether pure or mixed products, for example in effluents and discharges.

 These models generally facilitate the study of short-term or long-term cytotoxic effects and allow for viability tests, such as function tests (targeting a function or mechanism of the cell). ).

 These cell cultures, cells and biological material of the invention are also advantageously used in pathology. In particular, they constitute models of choice for highlighting the cytopathic effects of a virus, or other pathogenic organisms, on a culture, and for generally investigating the effects of these viruses and organisms on marine invertebrates. In another aspect, they also allow the production for research purposes of these pathogenic organisms.

 Also within the scope of the invention are the kits for carrying out these tests.

 These kits include the biological material and reagents needed to perform the tests.

 Suitably, the biological material comprises the isolated cells, the frozen cells, the culture media being provided in separate containers, or the cell cultures, with their culture medium.

 The invention also provides with cell cultures tools of great utility for performing in vitro fertilizations for hatchery applications.

 The use of frozen lots of oocytes, where appropriate spermotazoides, allows to have at any time reproductive cells.

 It is thus possible to release seasonal constraints and be assured of a constant quality all year long.

 Other features and advantages of the invention are given in the following description of experiments relating to two marine bivalves, the scallop and the oyster.

Reference is made to Figures 1 to 31 which represent
FIGS. 1 and 2, the autoradiographs of cultures of St. Jacques shell heart cells (respectively of myocytes and of epithelial type cells) after incorporation of 3H thymidine;
FIG. 3, autoradiography of scallop gill cell cultures, after incorporation of 3H thymidine;
FIGS. 4 and 5, a photo of a Brd U test on cultured St. Jacques shell cells after incubation of 48h and 24h, respectively
FIG. 6, the incorporation of leucine 14C and 3H thymidine (Dpm / μg prot) as a function of the culture time for shell core cells.
St Jacques,
FIGS. 7 to 9, autoradiography of St. Jacques shell heart cells after incubation of leucine 14C,
- Figure 10, the autoradiography of scallop gill cells after incubation of leucine 14C,
FIGS. 11A and B and 12, respectively the electrophoresis (11A) and the autoradiography of the electrophoresis (11B and 12) of the St-Jacques shell cell culture proteins, at different days of culture.
FIGS. 13 and 14, autoradiography photos of shell heart cells after incubation of 14C acetate,
FIGS. 15 and 16, the autoradiographs of thin-layer chromatograms separating lipids neosynthesized from 14C acetate by scallop core cells,
FIGS. 1A and 17B, the profile, respectively, of polar lipids and of neutral lipids, of heart cells of scallops, just after isolation and after 7 days of culture,
FIGS. 18A and 18B, histograms of fatty acid compositions of polar lipids and neutral lipids in these cardiac cells, just after isolation and after 7 days of culture,
FIGS. 19A and 19B, the quantitative fatty acid composition of polar lipids and neutral lipids in cardiac cells just after isolation and after 7 days of culture,
FIG. 20, the fatty acid composition of the base medium containing FCS,
FIGS. 21 and 22, autoradiographs of electrophoresis of neosynthesized proteins from 35S methionine by shell core cells
St Jacques;;
FIGS. 23 to 25, the autoradiographs of thin layer chromatograms separating lipids neosynthesized from 14C acetate by scallop core cells,
FIG. 26, the incorporation of leucine 14C and 3H thymidine, measured by scintillation counting, after 7 days of culture, by frozen-thawed cells, compared with cells of the same batch seeded in isolation,
FIG. 27, electrophoresis autoradiography of proteins neosynthesized from 35S methionine by shell core cells.
Scallops not frozen and frozen-thawed after 7 days of culture in the basal medium
FIGS. 28A and 28B, autoradiagraphy of thin-layer chromatograms separating lipids neosynthesized from 14C acetate by frozen and frozen frozen scallop core cells, frozen after 7 days of culture in the basal medium,
- Figure 29, the effect of mercury chloride (10-2M) on the oxygen consumption curve of the scallop shell gill cells in suspension;
FIG. 30, the effect of heavy metals on the oxygen consumption curve of oyster embryo cells in suspension
- Figures 31A to 31D, the effect of heavy metals on the ester activity of core cells of scallops in primary culture.

Materials and Methods Used - Results Achieved
The biological material, animals and cells, consists of
- For animals, scallops Pecten maximus and oysters Crassostrea gigas. Scallops are harvested by dredging, either in Baie de St-Brieuc or
Rade de Brest (Finistère). They are kept in hatchery for about a month before handling.

 Oysters for embryos are also packaged at the hatchery for controlled maturation for one month.

 - For cells, embryonic cells and differentiated cells.

Embryonic cells are obtained by dissociation of oyster embryos at the 416-cell division stage. Embryos are obtained by in vitro fertilization, proceeding as follows
- Removal of gametes
The method conventionally used in hatcheries to obtain, in bivalves, the artificial emission of gametes is induction by thermal shock, of variable amplitude according to the species (7-80C for the oyster).

 For the oyster, it is also possible to puncture the mature sex cells directly into the gonad.

 After opening the oyster, the genital gland is disinfected by painting an antiseptic (betadine) half diluted in seawater. The gametes are taken directly into the gonad by puncture with a sterile syringe. A light microscopic examination is performed to check whether they are oocytes or spermatozoa and to assess their quality (motility for spermatozoa, state of maturity for oocytes).

The male and female gametes are collected separately, in large volumes of sterilized seawater, filtered at 0.2 μm, and added with a solution of antibiotics (solution A given below)
- Fertilization
Cross fertilization is carried out according to the method of Gruffydd and Beaumont (1970) (the bibliographic references are given at the end of the description): the sperm of several males in a mixture is progressively added to the oocytes until five to seven days are observed. spermatozoa around each oocyte. The emission of the first polar body makes it possible to verify microscopically that the fertilization took place.

- Embryogenesis - Collection of embryos
The eggs are maintained in a sterile atmosphere at a temperature of 15 ° C until embryos at the development stage of 8-16 cells are obtained, which are then concentrated, under a laminar flow hood, by filtration on sieves. porosity 30 pin then aliquots.

Differentiated cells are isolated from various scallop organs
- mantle and labial palps, whose epithelia are supported by a thick connective tissue rich in fibroblasts,
the muscle and the heart, which make it possible to carry out cell suspensions free of microbial contaminants. In particular, the heart, protected by a pericardial membrane, is not in direct contact with seawater, which necessarily contains various types of microorganisms,
- Gills, organs largely in contact with seawater, constituting an interesting model for studies in toxicology.

I) CULTURE
The animals and tissues are first subjected to pre-treatment, then the cells are isolated and cultured.

These different steps are carried out as follows
1) - PRE-TREATMENT
The animals, before dissection, are carefully brushed in filtered seawater (at 0.2 μm) in order to get rid of their epifauna. Before opening the valves, the shells are disinfected (for example with alcohol 900).

 The scallops and oysters are then opened in a sterile atmosphere and washed with a large volume of sterile seawater and then disinfected. For example, half-diluted betadine is used in sterile seawater for 5 minutes. The different tissues are dissected sterile.

 Before being dissociated, the various tissues are treated with antibiotic baths of decreasing concentration (4X, 2X, 1X). Each bath lasts five minutes.

Advantageously, the solution of antibiotics A, corresponding to the following composition, is used, other solutions being however usable
Solution A
Penicillin - Streptomycin: (Gibco)
50 IU / ml-50 μg / ml
Kanamycin (Sigma): 10 μg / ml
Gentamycin (Sigma): 20 μg / ml
Antibiotics are dissolved in sterile seawater.

2) - ISOLATION OF CELLS
Two techniques are used to isolate cells: the explant technique and tissue dissociation.

A. by explants
The various organs removed and decontaminated are sterilely cut into fragments of about 1 mm3.

The samples obtained are deposited one by one on the culture medium and then gradually covered with medium. In order to eliminate the individual factor, each culture is made with fragments from several individuals.

 Cell migrations are observed from heart explants, gills and labial palps.

 The cells that migrate from the gill and heart fragments are of the fibroblast type. For the labial palps, epithelial cells are also observed.

 These cell migrations usually occur after two to three days of culture in the case of the heart and gills, later for the labial palps (about fifteen days). The number of cells migrating from the explants increases for two to three weeks, and in the case of the heart, they eventually form a cell layer, then they gradually detach from the bottom of the boxes and degenerate. The heart explants continent to contract rhythmically even after three weeks in vitro.

B. by dissociation of tissues
DISSOCIATION SYSTEM
Assays for enzymatic dissociation, chemical or mixed, were carried out for the various organs studied and for the embryos. Among the enzymes and products used, mention will be made of
pronase: Streptomyces griseus (Sigma)
- elastase: Porcine pancreas (Boehringer)
- trypsin: Bovine pancreas (Boehringer)
collagenase: Clostridium histolyticum (Sigma)
pepsin: Porcine stomach mucosa (Sigma)
collagenase dispase: Acinetobacter
Iophagus / Bacillus polymixa (Boehringer)
- EDTA: (disodium salt of ethylene diamine tetraacetic acid)
- ready-to-use non-enzymatic dissociation solution (HBSS Sigma)
Alsever, an anti-aggregating agent based on EDTA whose composition is as follows: glucose, 20.8 g; sodium citrate, 8 g; EDTA, 3.3 g; NaCl, 22.30 g; distilled water 100 ml.

 These compounds, used at varying concentrations in Hanks solution without calcium or magnesium, pH 7.3, whose osmolarity is adjusted to 1100 mOsm by addition of NaCl, are generally added to the mixture of antibiotics.

 The contact time of the cells with the dissociation solution is one hour. The treatment is completed by mechanical agitation. The experiments are performed at room temperature. The enzymatic action is stopped by two successive washes in sterile seawater.

 With the exception of the muscle for which several enzymes make it possible to obtain isolated cells with a viability rate of the order of 80% or more, the treatment of one hour at 150C with the pronase at 0.2% gives often the best results for the other organs studied. Average viability rates are close to or above 80%.

 Pronase, alone or in combination, is particularly suitable for dissociating embryos, well protected by a thick and resistant envelope.

 Electron microscopic examination of oyster embryonic cells, gill cells and scallop heart cells isolated by pronase confirms that the majority of the cells show no ultrastructural alteration.

CONTAMINATION OF CEILAR SUSPENSIONS
Some dissociated cell populations have been found to be heavily contaminated by bacteria, particularly gills and mantle.

 A decontamination protocol is then advantageously followed according to which a mucolytic product, dithiothreitol (DTT) is used.

 The tissues are immersed for 15 minutes in a sterile seawater bath containing 25 mM, 50 mM or 100 mM DTT. After washing with sterile seawater, they are then immersed in antibiotic baths of decreasing concentrations (4X, 2X, 1X), before being dissociated. Microbiological control is carried out by seeding the cell suspensions on Zobell medium (culture medium for marine bacteria).

 The action of DTT on the dissociated cells was also checked against control lots.

This study was done for the various concentrations of the product, for various pronase concentrations (0.1%, 0.05% or 0.025%) and for dissociation conditions of 12 hours at 40C or 15 minutes. at room temperature. The data are processed by variance analysis to compare the various results obtained.

 The bacteriological control of gills thus treated shows a marked decrease in the number of colonies. This decrease is "dose-dependent": the number of colonies decreases as the concentration of DTT increases.

 This treatment is reinforced if necessary by a more specific treatment with antibiotics.

 The viability of the gill cells is unaffected by such treatment, regardless of the dose of DTT.

 The use of 0.025% pronase supplemented with antibiotics for 12 hours at 4 ° C., after the action of DTT at 50 mM, gives the best results.

EVALUATION OF THE VIABILITY OF ISOLATED CELLS
The viability of the cells after isolation was evaluated by the Trypan blue exclusion method.

 Trypan Blue is a dye that penetrates only dead or damaged cells. This test had to be adapted to the marine environment by adjusting the osmolarity of the coloring solution using a low molecular weight sugar, arabinose, to avoid having too viscous solutions that would have hindered the sedimentation of the fish. cells.

 The counts are performed on cell suspensions diluted 1/20 in Trypan blue at 0.2% (w / v) in final concentration. The dead cells, stained in blue, and alive, uncolored, are counted by counting in Malassez cell, which allows the calculation of the cell viability rate.

3) - CULTURE OF CELLS IN THE MIDDLE OF
BASED
The cells cultured for these studies are essentially gill and heart cells of scallop shells and oyster embryonic cells.

CONDITIONS OF CULTURE
Enumeration and seeding of cells
After assessing the cell viability by the Trypan blue test, the cells are inoculated in a multiwell plate, in culture dishes or in flasks at the rate of 1.5 million viable cells per milliliter of medium.

Culture centre
Physicochemical parameters
a) Osmolarity
The osmolarity of the medium is adjusted to 1100 mOsm by addition of NaCl (Sigma). The measurements are made using an osmometer (Osmomat 030, Merck).

b) pH
The pH of the medium varies according to the tests from 7.3 to 7.9. A pH indicator, phenol red (Sigma), is added to the culture medium at a rate of 10 μg / ml to control for possible pH changes during cell culture.

c) buffer system
An organic buffer, Hepes (Calbochiem), is usually added to the culture media. Its final concentration is 10 mM.

The cultures of gill cells and scallop heart and those of oyster embryonic cells are carried out in an environment whose constitution is as follows
- sea water
- 10% Leibovitz L-15
- 10% fetal calf serum not
decomplemented
- Mixture of antibiotics: penicillin 50 IU / ml, streptomycin 50 μg / ml, kanamycin 10 μg / ml, gentamycin 20 μg / ml
- 10 mM Hepes
10 μg / ml of phenol red.

 The media are sterilized by 0.2 μm filtration and stored at -20 ° C until use. The serum is added extemporaneously.

Crop support
Plastic supports are used.

 The cells are cultured at 15 ° C and in an atmosphere not enriched with CO2.

 Whether for oyster embryos or for gills and scallop heart, cultured cells form, immediately after inoculation, small clusters, most of which adhere to the plastic. Two or three days later, many adherent cells, well spread on the surface, are visible near the aggregates. Their number increases according to the culture time, regularly up to 20 days. The cells then form a quasiconfluent carpet.

 For gill cells and embryonic cells, some cell clusters remain in suspension and intense ciliary activity is observed for about a week. In both cases, the adherent cells are of the fibroblast type.

In contrast, with respect to cardiac cell cultures, two types of adherent cells are observed after three or four days of culture.
epithelial-type cells, which are still in the minority,
fibroblast-type cells, the most numerous.

 For these cultures, terminal cell differentiation is observed after approximately one week of incubation: when the cells are quasiconfluent, they reconstitute symplasts, myotubes type multiinucleate formations, which contract at regular intervals of time.

Cell adhesion was evaluated as follows:
After three or four days of incubation, an MTT test and a deoxyribonucleic acid (DNA) assay are performed in order to compare the effectiveness of the different coatings with respect to the culture plastic. The amount of DNA makes it possible to assess the number of adherent cells, but it does not take into account the cell viability aspect, which is provided by the MTT test.

 The cells' DNA is determined by fluorimetry (Labarca and Paigen, 1980), after several washings of the culture wells. This assay utilizes the Hoechst reagent which, bound to the DNA, fluoresces at 458 nm when excited at 356 nm.

Incubation conditions
Taking into account hatchery conditions for oysters and scallops in a hatchery, a temperature of 15 C-16 C is used to cultivate the heart and gill cells of the Saint shell.
Jacques and a temperature of 20 C for the oyster embryonic cells.

 The cells are cultured in a normal atmosphere that is not enriched with CO2.

Renewal of the environment
The first change of medium is made after three to four days, once the cells have adhered well to the culture medium. Only half of the medium is renewed. Subsequently, a complete change of the medium is made every three days.

DETERMINING THE PERCENTAGE OF MEMBERSHIP
Four days after seeding, the adherent cells are, after washing in seawater, detached from the support by pronase at 0.025 g. After three to four minutes of treatment, they are taken up in a large volume of seawater, centrifuged at 1500 rpm for four minutes, and then counted in Malassez cell. The ratio between the number of adherent cells and the number of total cells seeded, multiplied by 100, gives the percentage of adhesion.

IDENTIFICATION OF CELLS ADAPTED IN CULTURE
Electron microscopy
In order to identify the adherent cells in culture, the cells were examined by transmission electron microscopy.

For the heart of the scallop shell,
in the cytoplasm of almost all fibroblast cells the presence of myofibrils is noted. Their examination proves that they are cardiomyocytes. The cytoplasm is usually loaded with lipid inclusions, compared to that of the heart cells before seeding.

immunocytochemistry
The fibroblast cells adhering to the support were characterized by indirect immunofluorescence, using primary antibodies, antimyosin Rb (Sigma M-7648) or anti-tropomyosin Rb (Sigma T-3651) diluted at 1 / l0ème. After brief washing in phosphate buffer (PBS - osmolarity 1100 mOsm), the cells are fixed for 30 minutes at 4 ° C. with 4% paraformaldehyde in 0.1M sodium cacodylate buffer, pH 7.4, which are then washed. twice for 15 minutes in 0.1M cacodylate buffer, then incubated in three 30-minute baths of NH4C1 to saturate the aldehydes.A 30-minute treatment in serum albumin of beef (BSA) with 3% saturates non-specific sites.

 The cell membrane is permeabilized by incubation for one hour in 0.1% saponin in PBS buffer. The first anti-myosin antibody or antitropomyosin diluted 1 / 10th in PBSsaponin 0.1% is then applied for 1 hour. After two washes with the PBS-saponin mixture, the cells are incubated in the presence of the second antibody, antirabbit IgG-FITC conjugate (Sigma F-0511), diluted 1 / 50th for one hour. They are then washed twice for 30 minutes with PBS and slats are applied on the bottom of the boxes. The assembly is carried out with PBS-glycerol (lv / 9v). The observation is made under a fluorescence microscope. Negative control boxes not treated with the first antibody or the second antibody are run in parallel.

 Fusiform heart cells, adherent in culture, observed under a fluorescence microscope after incubation in the presence of anti-myosin antibody, marker of muscle fibers, and then a secondary antibody coupled to fluorescein, appear green fluorescent. The negative control, which is not treated with the primary antibody, has only a few fluorescent intracytoplasmic inclusions. These inclusions are likely to be lipofuschins or lipocarotenes.

 A strongly positive reaction is also noted after treatment with anti-tropomyosin antibody, which is less specific for muscle cells than myosin. In this case, the tropomyosin filaments are clearly visible in the cells. The controls, not incubated in the presence of primary antibody, have only a few yellow fluorescent globules.

 It is interesting to note that antibodies of mammalian origin react positively with the bivalve cells, but it is necessary to use them at higher concentration.

 These various observations confirm that most of the adherent cells of St. Jacques shell heart, of fibroblastic appearance, are myocytes.

 Crop quality and functional capacity of the cultured cells are evaluated by microscopic observation and using functional capacity tests for microscopic observation, using an inverted microscope, taking into account the following criteria: cell adhesion, spreading, presence or absence of intracytoplasmic vacuoles, general granular appearance. A more specific study in electron microscopy also made it possible to check the cell integrity.

 For functional capacity tests, autoradiography techniques, the use of a thymidine analogue, 5-bromo2'-deoxyuridine (BrdU), scintillation counting, electrophoresis and electrophoresis autoradiography, thin layer chromatography (TLC) and chromatogram autoradiography.

a) - Autoradiography
The incorporation of radiolabeled precursors into the cells in culture is visualized in order to specify the places of synthesis of the macromolecules. After labeling the cells, a photographic emulsion sensitive to radioelements and containing a silver salt is cast on the cultures; the radioelements impress it, which results in black metallic silver grains. We thus study
the neosynthesis of DNA by the use of 3H thymidine
the neosynthesis of proteins by incorporation of leucine 14C
the neosynthesis of lipids using 14C acetate
The location of silver grains inside the cell can be facilitated by histological staining to differentiate the nucleus and cytoplasm.

The different stages of this technique are as follows
adherent cells in culture;
- 2 quick washes in seawater
- incubation: with marked amino acid
- rinses (7 x 5 min) with sea water;
fixation: 3 min with 2.5% glutaraldehyde in 0.1M Na cacodylate buffer;
washing: 2 x 5 min with 0.1M Na cacodylate buffer;
- elimination of the buffer
- casting a photographic emulsion;
- exhibition: 1 week
- revelation: 4 min
- washing with distilled water
- staining (MGG or nuclear red).

The cells in culture are rinsed twice with sterile seawater and then incubated for 24 hours in the presence of 14C leucine at 0.2 pCi / ml (specific activity: 311 mCi / mol, Amersham), 14C acetate at 3 μCi / ml (specific activity: 56 mCi / mol,
Amersham) or 3 H-thymidine at 1 μCi / ml (specific activity: 27 mCi / mol, Amersham) in artificial seawater containing antibiotics.

 After incubation, the cells are rinsed seven times with seawater, in order to eliminate non-specific radioactivity; a count is made on the last supernatant to verify in liquid scintillation that no more radioactivity remains.

 Various witnesses not treated by the marked elements undergo the same treatments. The cells are then fixed to 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 1100 mosm for three minutes on ice. They are rinsed twice with cacodylate buffer and can then be stored in this buffer at 40C, until further processing.

 After removal of the buffer, an emulsion (Amersham LM-1) is poured onto the culture dishes.

After about a week of exposure to 40C, the autoradiographs are developed: the revelation is carried out for 4 minutes at room temperature (developer II strong Hypam). The culture dishes are washed with distilled water and kept in distilled water until stained by a May-Grunwald.
Giemsa "(MGG), ie nuclear red.

b) - BrdU test
To detect a possible proliferation of the cells in culture, a technique using a thymidine analog, BrdU (RPN-Amersham kit) is used. BrdU is incorporated into the replicating DNA, and then localized using a nuclease-coupled specific monoclonal antibody (anti-BrdU) that denatures the DNA. To increase the sensitivity of the technique, cells are simultaneously exposed to
BrdU and 5-fluoro-2'-deoxyuridine, a thymidilate synthetase inhibitor that increases BrdU incorporation by decreasing competition with exogenous thymidine. The detection of the primary antibody is carried out using the IgG2A mouse antibody and then coupled to the peroxidase-antiperoxidase system. This test has the advantage of not using a radioactive element.

We operate according to the following steps
adherent cells in culture
incubation: BrdU (24 h);
- fast wash in PBS
fixing: acetic acid-ethanol (30 min);
- washes with PBS (3 x 3 min)
- incubation: PBS + 10% FCS (saturates the sites
nonspecific)
- incubation: nuclease-anti BrdU (1 h)
washing with PBS (3 x 3 min);
incubation: anti-mouse-peroxidase IgG2A (30 min);
washing with PBS (3 x 3 min);
- coloring: DAB + "intensifying substrate" (10 min);
- GIEMSA staining (3 min).

 BrdU is diluted 1/1000 in the culture medium. The cells are incubated for 24 to 48 hours in the presence of this substrate and then washed with PBS (osmolarity adjusted to 1100 mOsm).

 They are then fixed with acetic acid ethanol for 30 minutes at room temperature (absolute ethanol 90%, glacial acetic acid 5%, water 5%).

 Three washes of three minutes each are carried out in PBS before saturating the aspecific sites with PBS supplemented with 10% FCS.

 Three phosphate buffer washes are then performed. The reconstituted nuclease / anti BrdU mixture is then contacted with the cells for one hour at room temperature.

 Three new washes in PBS (3 minutes each) preceding the application of IgG2A antisourisperoxidase for 30 minutes at room temperature are carried out.

 After three more washes in PBS (3 minutes each), a concentrated aliquot of diethylaminobenzidine (DAB) is diluted in 50 ml of phosphate buffer, to which five drops of intensifying substrate are added.

 The coloration lasts ten minutes. It is followed by three washes with distilled water, three minutes each.

 GIEMSA staining is then performed.

 In cells that have incorporated BrdU, the nuclei and especially chromatin are stained dark blue. Culture dishes, "negative controls", are processed without applying the BrdU-antiBrdU system (replaced by PBS).

c) - Scintillation counting
Measurement of the incorporation of 3H thymidine and leucine 14C makes it possible to assess the capacity of the cells in culture to neosynthesize DNA and proteins.

Typically, the cells are subjected to the following steps
adherent cells in culture
- 2 rapid washes: sea water;
incubation: 24 hours with labeled amino acid;
- washes in seawater (2 x 5 min)
sonication in 150 μl PBS; followed by
protein assay (Bradford) over 50, ul and
assay of the incorporated radioactivity on 100 μl to which 250 μl of trichloroacetic acid (TCA) 60% + 250 μl of BSA is added (precipitation: 1 night at 4 ° C., centrifugation: 3000 rpm 15 min, taken up by
TCA 10%; centrifugation: (3000 rpm 15 min); take up by TCA 5% (x 2); centrifugation (3000 rpm / min); addition of formic acid, measurement of radioactivity in liquid scintillation).

 The adherent cells are rinsed twice with sterile seawater, and then the cultures are incubated for 24 hours in the presence of leucine 14C 0.2, uCi / ml (specific activity: 311 mCi / mmol, Amersham) or thymidine 3H at 1 μCi / ml (specific activity 27, μCi / mmol, Amersham) in sterile artificial seawater supplemented with antibiotics. They are then rinsed twice in seawater and then frozen "dry" at -80 ° C.

Precipitation of proteins and counting of radioactivity
The cells are sonicated in 150 of PBS. Fifty microliters are taken for the determination of proteins by the technique Coomassie blue, the 100 pl "remaining" used to measure the incorporated radioactivity. To these 100 μl are added 250 μl of trichloroacetic acid (TCA) (Carlo Erba) at 60% and 250 μl of serum albumin of beef (Sigma) at 0.6 mg / ml. The precipitation is overnight at 40C. The precipitates are pelleted by centrifugation at 3000 rpm for 15 minutes. The supernatants are removed and the pellets are taken up in 250 μl of 10% TCA. The precipitates are then washed twice with 5% TCA in order to remove the unincorporated precursor; 150 μl of formic acid are then added to dissolve the proteins and the radioactivity is measured by liquid scintillation.

Protein Assay with Coomassie Blue, by Bradford Method (1976)
A standard range of serum albumin from beef is made from a stock solution at 0.25 mg / ml.

Five successive one-half dilutions are carried out in PBS buffer. Fifty microliters of each sample to be analyzed and the standard range are duplicated in a 96-well reading plate.

After adding 200 μl of Coomassie Blue R-250 (Biorad) diluted 1/4 in ultrapure water (Milli Q filtration), the absorbance is measured by spectrophotometer at 620 nm.

- Calculation of incorporated radioactivity
The counts obtained using the ss counter (Packard) are then reduced to the amount of protein per culture well. The results are expressed in
Dpm / μg of protein (Dpm = disintegration per minute).

d) Electrophoresis and autoradiography
Electrophoresis
In order to visualize possible differences in protein synthesis as a function of culture time, these compounds are separated by electrophoresis after incubation of the cells with 35S-labeled methionine.

The steps implemented are as follows
adherent cells in culture
- 2 quick washings: seawater
incubation: 24 hours with 35S methionine;
- washings in seawater (2 x 5 min);
- dry freezing at - 80 ° C;
- "sonication": dissociation solution (2 x 20 sec);
heating: 100 ° C, min;
- liquid scintillation counting on a sample
deposition on electrophoresis gel (migration, silver nitrate staining, gel drying: 370 ° C., 12 hours, film deposition: autoradiography and, exposure-80 ° C., revelation).

Incorporation of labeled methionine
After two rinses in sterile seawater, the cells are incubated for 24 hours in the presence of 35 S methionine at 5 uCi / ml in sterile artificial seawater supplemented with antibiotics. After incubation, the cells undergo two washes in seawater then they are frozen "dry" at -800C.

Obtaining proteins and counting
The cells are "sonicated" (2x20 sec) in 250 μl of 10mM Tris-HCl; 1mM EDTA, pH 8 sodium dodecyl sulfate (SDS) 2.5%; mercaptoethanol 5%; bromophenol blue 0.01%. The resulting suspensions are then heated at 1000C for five minutes. A sample is taken and the radioactivity is counted on the liquid scintillation counter.

Electrophoresis and staining
Protein separation is carried out by polyacrylamide gel electrophoresis in the presence of
SDS (Biorad) according to the method of Laemmli (1970).

The gel used is a 10-15 gradient polyacrylamide gel. Electrophoresis is performed using the Phast System (Pharmacia). The samples are diluted to obtain the same amount of radioactivity. One microliter of each sample is deposited on the gel. After migration, the gel is stained with silver nitrate according to a method derived from that of
Heukeshoven and Demick (1985). The gel is then dried in an oven for 12 hours at 37 ° C.

Revelation
Films for autoradiography (hyperfilm ss max, Amersham) are deposited on the electrophoresis gels. The exposure is made at -80 ° C. for a variable time depending on the manipulations, depending on the amount of "deposited" radioactivity.

 The estimation of the molecular weight of the various proteins is carried out by reference to known molecular weight standards deposited at the same time as the samples: standard SDS (Biorad) Low molecular weight: 14 to 97 kilodaltons. High molecular weight: 40 to 200 kilodaltons.

e) - thin layer chromatography (TLC) and chromatogram autoradiography
In order to study lipid synthesis by cardiac cells as a function of culture time after incorporation of a precursor, 14C-labeled acetate, these are extracted. The various types of neosynthesized lipids are separated by thin layer chromatography and then revealed by contact with a film for autoradiography. In parallel, the lipids of the cardiac tissue are extracted and separated in the same way as for the cells in culture in order to try to identify the lipids present in the tissue and to compare them with those neosynthesized by the cells in culture.

The steps followed are diagrammed below
adherent cells in culture
- 2 rapid washes: sea water;
incubation: 24h with 14C acetate;
- washings in seawater (2 x 5 min);
- freezing at - 800C (at least 15 min)
- sonication: methanol
- chloroform
- stirring 2 min, then addition of Kcl
stirring: 2 min, centrifugation (2000 rpm for 5 min)
- organic phase recovery;
- evaporation under nitrogen;
chloroform - methanol (2/1) and
- liquid scintillation counting on
a sample, and
- Silica plate deposit on the remaining part (migration, film deposition autoradiography, exposure -800C, revelation).

Incorporation of marked acetate
The cells are rinsed twice with sterile seawater, then the cultures are incubated for 24 hours in the presence of a 14C acetate solution at 5 μCi / ml (specific activity 56.2 mCi / mmol, Amersham). in sterile artificial seawater with antibiotics. After incubation, the cells are rinsed twice with seawater and then dry-frozen at 80 ° C.

Extraction and counting of lipids
Lipids are extracted according to the technique of
Bligh and Dyer (1959). The cells are sonicated (2 × 20 sec) in 1 ml of methanol and then transferred to glass tubes. Three ml of chloroform are added and the tubes are then stirred for two minutes by vortexing. After addition of 1 ml of
KCl, stirring for two minutes vortexing and waiting for two minutes, the tubes are centrifuged at 2200 rpm for five minutes. Two phases are obtained. The organic phase is recovered and evaporated under nitrogen. The residues are taken up in a chloroform-methanol mixture (2-1). An aliquot fraction is taken and the radioactivity is measured by liquid scintillation counting.

Separation of lipids by thin layer chromatography
Samples of 5 to 20 μl depending on the number of strokes (approximately 10,000 Cpm) are deposited on 60WF 2545 silica plates. Two migration systems are used.
For the separation of neutral lipids: the migration is done in a mixture hexane - diethylétheracide formique (90/10/1) on 12 cm, according to the technique of Oshima and ai. (1987).

 By separation of the polar lipids: the first migration lasting ten minutes is carried out in acetone, then a second migration is carried out in a mixture chloroform-methanol-water (65/35/4) on 12 cm, according to the technique of Ratnayake and Ackman (1985).

Revelation
Films for autoradiography (hyper film ss max, Amersham) are deposited on the chromatograms for exposure at -80 C for a variable time depending on the amount of deposited radioactivity (Cpm).

 An identification of the different lipids is attempted thanks to unmarked standards, which migrate at the same time as neosynthesized and radiolabelled lipids.

The standard products (Sigma) used are the following
For neutral lipids
palmitic acid,
- cholesterol,
- cholesterol palmitate,
- tristearin,
1,2-Oleylglycerol.

For polar lipids
- Lysophosphatidylcholine
Lysophosphatidylethanolamine
- phosphatidylcholine
phosphatidylethanolamine.

 Tristearin and 1,2-oleyl glycerol are revealed by sputtering 2 ', 7' dichlorofluorescein (Sigma, diluted 1/4 in ethanol). The spots are then visualized under UV.

 The other unmarked standards are revealed by immersing the plates for 20 seconds in a water-acetic acid-sulfuric acid solution (95/5 / 0.5) containing 0.5% of copper sulfate and then heating at 1500C for 15 minutes and at 200 ° C during this time.

 The intensity of the spots corresponding to the synthesized lipids is visually appreciated on the autoradiography films.

f) - Fatty acid composition of the cells by gas chromatography
A fatty acid analysis of the heart cells is performed to compare their profile in the cells after isolation and after seven days of culture in the basal medium. The cells, after counting, are taken up in chloroform-methanol (2/1). This extract is then evaporated under vacuum, taken up in a small volume of chloroform-methanol (98/2) and the neutral lipids (triglycerides, free sterols and sterol esters) and polar lipids (phospholipids) are separated on a microcolumn of silica (30). mm x 5 mm) using respectively CHCl3-MeOH (98/2) and MeOH. The fractions are collected in tip tubes containing C23: 0 (internal standard) and BHT (Butylhydroxytoluene-antioxidant). The fatty acids are then transesterified with methanol in the presence of
14% BF3 (Metcalfe and Schmitz, 1961) and the methyl esters of the fatty acids obtained are analyzed on a DBWAX capillary column (25 mm × 0.32 mm, 0.2 μm epf thickness of the phase film). Fatty acids are identified by comparing their retention time with that of standards. The fatty acids are named according to the formula C: X (nY) where C is the number of carbon atoms, X is the number of double bonds and Y is the position of the first double bond counted from the terminal CH3. An analysis of the fetal calf serum was also performed according to the same protocol.

BIOSYNTHETIC ACTIVITY OF EH CULTURE CELLS
The results obtained with cells in culture of the heart and scallop gills are reported below, as a function of the culture time, in the base medium and then in a supplemented medium.

- IN THE BASIC ENVIRONMENT
OSYNTHH: SE DNA Histoautoradiography
As shown in Figures 1 and 2, the core of some core cells cultured for seven days, after incorporation of tritiated thymidine, contains many silver grains (see arrow). This labeling is observed both for the myocytes (Figure 1) and for the epithelial type cells (Figure 2).

 These results highlight the synthesis of DNA by the cells in culture.

 Similarly, silver grains are observed in the gill cell nucleus (Figure 3), proving again that these cells neosynthesize DNA.

5-bromo deoxyuridine labeling (BrdU)
On 7-day heart cultures, it is observed that after incubation in the presence of BrdU, the nucleus of certain cells is colored black (FIGS. 4 and 5). This positive reaction also proves that heart cells synthesize DNA. The number of positively labeled nuclei varies with the incubation time of the cells with BrdU. Indeed, after 48 hours of contact with the product (Figure 4), the number of black nuclei is much greater than after a 24-hour incubation (Figure 5).

Some images also evoke mitoses (Figure 5).

Scintillation counting
Scintillation counting of 3H thymidine incorporation versus culture time, the results of which for three different cultures are grouped together in Figure 6, indicate that thymidine is incorporated. But this incorporation is weak. The values found at the beginning of culture are also of the same order of magnitude as those recorded for rat hepatocytes, taken as a control, in order to validate the technique. An increased synthesis of DNA is noted after one week of culture even if the peak of synthesis does not appear strictly the same day according to the cultures.

OSYNTHZSE OF PROTE1VES
- Histoautoradiography
The cytoplasm of many cultured heart cells is intensely labeled after incorporation of 14C leucine as shown in FIGS. 7-10. These figures respectively relate to nuclear red-stained fibroblastic cells, MGG-stained fibroblast cells and epithelial cell plaques. intensely marked by leucine 14C. Silver grains are also indicated by an arrow.

Cardiomyocytes (FIGS. 7 and 8) and epithelial-type cells (FIG. 9) react positively, demonstrating their protein synthesis.

 A similar observation is made for cultured gill cells (Figure 10).

 It is observed that the silver grains in the cytoplasm of the cells after the incorporation of leucine 14C. These cells also neo-synthesize proteins.

- Electrophoresis and autoradiography
Electrophoresis
The electrophoresis and electrophoresis autoradiographs, obtained at different culture times, after incubation of the heart cells with 35S methionine, are grouped together in FIGS. 11 and 12.

 The electrophoresis autoradiography shows that the cells neosynthesize a large number of proteins, for all the culture times studied, and that the profiles are the same (FIG. 11B). In comparison with the electrophoresis carried out, the low and high molecular weight standards (designated by BPM and HPM in the figures) being deposited in parallel, it appears that a protein with a molecular weight of approximately 45 kilodaltons is still predominantly neosynthesized (see arrow).

 Electrophoresis reveals the presence of a protein with a molecular weight of about 66 kilodaltons (see arrow).

 The protein mainly present in the cells does not correspond to that which is neosynthesized in greater quantity. This protein is likely to be rapidly reused or degraded.

 Figure 12 shows the electrophoresis autoradiography (lane 1: after 27 days of culture, lane 2: after 29 days of culture). It is found that cells continue to neosynthesize proteins even after 29 days of culture.

- Scintillation counting
For the cultures where the counts have been carried out, it is noted that the incorporation of leucine is more or less intense as a function of the culture time. A first peak of protein synthesis is noted after 7 days of culture in all three cases (Figure 6). A second peak is then systematically observed but it appears more or less later, after 15 to 24 days depending on the crop.

An increased synthesis of proteins therefore appears to occur in successive waves,
CSYNTHESIS OF LIPIDS Histoautoradiography
Figures 13 and 14 show heart cells respectively stained with MGG and nuclear red after incorporation of 14C acetate. It is noted that the silver grains are essentially located at the level of the cytoplasmic membrane (see arrow).

 Similar observations are made for gill cells.

Thin layer chromatography and chromatogram autoradiography
Figures 15 and 16 show autoradiographs of thin layer chromatograms obtained after incubation of the cells with 14C acetate, after different culture times.

 Autoradiographs show that cells in culture neosynthesize, regardless of the culture time, many lipids, but in varying proportions; Polar lipids are synthesized in greater quantities than neutral lipids.

 For neutral lipids, at least 4 types of lipids can be distinguished, labeled with letters from A to D depending on their migration distance from the deposition (Figure 15).

 With regard to polar lipids, at least 12 different types are neosynthesized by the cells in culture; they are also indicated by the letters A to L (Figure 16).

 If we compare the lipid profiles obtained as a function of the culture time, no major difference is observed up to 19 days. After 19 days of culture, slight quantitative and / or qualitative changes in neutral lipids such as polar lipids are noted by comparing the chromatogram autoradiographs obtained for the same culture. For neutral lipids, one of the lipids (C) is synthesized in smaller amounts while another lipid (B) appears (see arrow). With regard to polar lipids (lipids 16), a new lipid is synthesized (E) (see arrow) while one of them (F) (see arrow) seems to have disappeared and another ( I), is synthesized in larger quantities.

Analysis of fatty acid composition of heart cells
The fatty acid composition of the heart cells immediately after isolation (Jo) was compared, by gas chromatography, to that of the cells cultured for seven days in the basal medium containing normal FBS (Figures 17, A and 17 B). ), SVF whose fatty acid profile has been established.

An increase in lipids in the cells after seven days of culture is noted, the cells containing 0.16 g of fatty acids / ug of protein after isolation against 0.9 ug of fatty acids / ug of protein after seven days of culture. The amount of phospholipids is multiplied by about three (0.127 μg / μg of Jo proteins against 0.39 μg / μg of
J7), that of the neutral lipids by 17 (0.03 μg / μg of Jo proteins against 0.51 μg / μg of protein on day 7).

 There is therefore in cells a change in lipid compartments after seven days of culture in the basal medium containing FBS.

 The histograms shown in Figures 18A and 18B show the difference in fatty acid composition, (expressed as a percentage), polar lipids and neutral lipids in Jo cells and after seven days of culture.

 Whether for polar lipids or neutral lipids, the percentage of saturated fatty acids remains generally the same; that of monounsaturates increases after seven days of culture. The percentage of fatty acids of the series (n-6) increases while that of the series (n-3) decreases.

 Figures 19A and 19B show the quantitative fatty acid composition of polar lipids and neutral lipids after isolation and after seven days of culture.

 If we compare the fatty acid profiles of cells after seven days of culture with the fatty acid profile of FCS (Figure 20), we can see that the fatty acid profile of the cells cultivated in the presence of FCS reflects the composition of this one. Accumulation in 20: 4 (n-6) and 20: 5 (n-6) cells was observed as well as selective uptake of 22: 6 (n-3).

- IN THE COMPLEMENTING ENVIRONMENT
Measures of incorporation of labeled precursors make it possible to highlight the role of certain factors of mammalian or marine origin.

 Thus, incorporation measures of 14C leucine and 3H thymidine were carried out by the cells cultured in the variously complemented base medium. These measurements, made to evaluate the effect of each factor added to the medium, were made only between five days and nine days of culture, given that for cells cultured in the basal medium a first peak of Protein is observed at 7 days.

 The results obtained are collated in the following table.

BOARD
Effect of the various complements tested on the
DNA and protein synthesis

<tb><SEP> J5 <SEP> J7 <SEP> J9
<tb> Haemolymph <SEP> of oyster <SEP> + <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> + <SEP> + <SEP> Synthesis <SEP> DNA
<tb> Extract <SEP> of oyster embryos <SEP><SEP> + <SEP> - <SEP> + <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> + <SEP> - <SEP> Synthesis <SEP> DNA
<tb> EGF <SEP> - <SEP> - <SEP> + <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> - <SEP> - <SEP> Synthesis <SEP> DNA
<tb> haemolymph <SEP> of <SEP> shell <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> - <SEP> - <SEP> Synthesis <SEP> DNA
<tb> Extract <SEP> of embryos <SEP> of <SEP> shell <SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> + <SEP> - <SEP> Synthesis <SEP> DNA
<tb> Extract <SEP> of <SEP> ganglia <SEP> from <SEP> mold <SEP> - <SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> + <SEP> - <SEP> Synthesis <SEP> DNA
<tb> Extract <SEP> SKC <SEP> 5 <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb> (medium <SEP> with <SEP> SVF <SEP> delipidated) <SEP> + <SEP> - <SEP> Synthesis <SEP> ADN
<tb> Extract <SEP> lipids <SEP> from <SEP> gonad <SEP> from <SEP> shell <SEP> + <SEP> + <SEP>#<SEP><SEP> Synthesis <SEP> proteins
<tb> (medium <SEP> with <SEP> SVF <SEP> normal) <SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> ADN
<tb> bFGF <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb><SEP> + <SEP> - <SEP> - <SEP> Synthesis <SEP> DNA <SEP>
<tb> Extract <SEP> SKC2 <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb> (medium <SEP> with <SEP> SVF <SEP> delipidated) <SEP> - <SEP> - <SEP> Synthesis) <SEP> DNA
<tb> Extract <SEP> of <SEP> lipids <SEP> from <SEP> Skeletonema <SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb> costatum <SEP> (medium <SEP> with <SEP> SVF <SEP> normal) <SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> DNA
<tb> extract <SEP> of <SEP> lipids <SEP> from <SEP> gonad <SEP> from <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb> shell <SEP> (medium <SEP> with <SEP> SVF <SEP> delipidated) <SEP> - <SEP> - <SEP> Synthesis <SEP> ADN
<tb> HGF <SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> proteins
<tb><SEP> - <SEP> - <SEP> - <SEP> Synthesis <SEP> DNA
<tb> positive effect: +, no effect:
.NEOSYNTHESIS OF PROTEINS BY AUTORADIOGRAPHY
ELECTROPHORESIS
Figures 21 and 22 combine electrophoresis autoradiographs showing neosynthesized proteins from 35S methionine by heart cells of scallops shelled in various complemented media.

In FIG. 21, the tracks T, 1, 2, 3, 4 respectively correspond to
T - Base medium with serum of veal (SVF) delipidated 1 - Base medium with added delipidated calf serum
lipid extracts from shell gonad
St-Jacques 2 - Base medium with delipidated veal serum
added lipids extracted from microalgae (2 μg / ml) 3 - Base medium with added delipidated calf serum
of lipids extracted from microalgae (5, ug / ml) 4 - Base medium with normal SVF
In FIG. 22, the tracks T, 1, 2, 3, 4, 5 respectively correspond to
T - Medium 1 - Medium supplemented with lipids extracted from shell
St-Jacques 2 - Medium supplemented with lipids extracted from Skeletoma
costatum 3 - Medium supplemented with ganglion extract
mold brain 4 - Medium supplemented with a mixture of vitamins,
antioxidants, growth factors 5 - Medium supplemented with HGF
The results obtained for the various media tested do not show any difference qualitatively in the profile of neosynthesized proteins.

NEOSYNTHESIS OF LIPIDS BY AUTORADIOGRAPHY
CHROMATOGRAM
FIGS. 23A and 23B, 24A and 24B and 25A and 25B show the autoradiographs of thin layer chromatograms, made after incubation of the cells with 14C acetate, after 7 days of culture, in the medium of base supplemented variously (Figures 23A and 23B) and in the base medium supplemented with marine lipids (Figure 24A and 24B).

 Figures A correspond to neutral lipids and Figures B to polar lipids.

In FIGS. 23A and 23B, tracks 1, 2, 3, 4, 5 and 6 respectively correspond to
1- Cells in culture in the basic medium
2- Cells grown in the basal medium supplemented with HGF
3- Cells in culture in the additive medium of vitamins, antioxidants, growth factors
4- Cells cultured in the medium supplemented with a mold ganglion extract
5- Cells cultured in the basal medium with added lipids extracted from Skeletonema costatum
6- Cells in culture in the basic medium supplemented with lipids extracted from gonad of Pecten maximus
In FIGS. 24A and 24B, tracks 1, 2, 3, 4 and 5 respectively correspond to
1- Control culture in the base medium supplemented with delipidated calf serum (FCS)
2- Cells grown in the additive medium of lipids extracted from St-Jacques shell gonad
3- Cells grown in the medium supplemented with lipids extracted from microalgae (5 μg / ml)
4- Cells cultivated in the medium supplemented with lipids extracted from microalgae (2 μg / ml)
5- Culture in basic medium with normal SVF
The cells synthesize at least three different classes of neutral lipids and eight classes of polar lipids.

Among the factors tested, including lipids, whether added to the medium supplemented with calf serum delipidated or not (Figure 24 A and Figure 24
B), only the extract of St-Jacques shell embryos modifies the profile of neo-synthesized lipid compounds.

Indeed in the category of neutral lipids, designated A and B on the autoradiographs appear specifically in the medium supplemented by this extract. In addition, the so-called type D lipid is neosynthesized in greater quantity than for the other factors studied. With regard to polar lipids, differences are also noted since two lipids, called C and F, are observed only in this case. On the other hand, if the lipid profile is only rarely modified, the intensity of the spots varies from one crop to another (Figures 23 and 24).

II FREEZING / DEFROSTING
-CONGELATION OF ISOLATED CELLS
Leibovitz L-15 medium supplemented with 10% FCS (osmolarity adjusted to 1100 mOsm) is used as a freezing and thawing medium.

 Six million enzymatically isolated cells are suspended in freeze ampoules in 0.8 ml of Leibovitz L-15 medium (GIBCO) supplemented with 20% FCS (D. Dutscher). An equivalent volume of Leibovitz medium containing the cryoprotectant to be tested is then introduced slowly into the cell suspension with gentle stirring.

 The cryoprotectant consists of dimethylsulfoxide (DMSO). This compound has the advantage of preventing the diffusion of cations and cytoplasmic components through the membrane pores closed off by the ice crystals.

 In order to prevent the release of heat produced by the addition of DMSO to the medium destroying the cells, the addition of the cryoprotectant is carried out very gradually.

 The percentage of DMSO is 12% and the contact time is 10 minutes.

 Blisters are filled with the cells in their freezing medium in the presence of DMSO.

 In order to slowly lower the temperature, the ampoules are packaged in several thicknesses of absorbent paper, transferred to a freezer and then to -800C for 12 hours, before being immersed in liquid nitrogen.

- DEFENSE
The thawing step is performed quickly to avoid the formation of intracellular crystals that would damage the cell.

 Typically, the samples are placed in a water bath at 37 ° C., and the freezing medium is diluted in medium containing 10% FCS as soon as the last ice crystals disappear.

 The cells, thus resuspended, are centrifuged at 1500 rpm for four minutes. The supernatant is removed and the cells are again resuspended in the culture medium. After counting with Trypan blue, the cells are inoculated at a rate of 1.5 million viable cells per ml of medium.

 The epithelial and fibroblast type cells are observed after freezing and thawing. The number of adherent cells increases as a function of the culture time. After 20 days of culture, the cardiac fibers also reconstitute myotubes when quasi-confluent mats of cells are obtained.

 The functional capacity of the frozen-thawed cells is assessed by the same criteria as those described for the cells seeded immediately after isolation: neosynthesis of DNA and proteins by scintillation counting, protein neosynthesis by electrophoresis autoradiography and lipid neosynthesis by thin layer chromatogram autoradiography. These studies are performed after seven days of culture compared to cells that have not been frozen.

- NEOSYNTHESIS OF PROTEINS AND DNA BY
SCINTILLATION COUNTING
The results of the incorporation of 14C leucine and 3H thymidine, measured by scintillation counting after seven days of culture by frozen frozen cells compared to cells of the same batch seeded in isolation are shown in Figure 26. In this figure Cf , C.cl, Cc2, Cc3 have the following meanings
Cf: fresh cells
Cc1: frozen cells: 10 min of contact
with DMSO
Cc2: frozen cells: 15 min of contact
with DMSO
Cc3: frozen cells: 20 min of contact
with DMSO
Cells which have been frozen incorporate both types of radiolabeled precursors as non-frozen control cells. The incorporation of leucine for frozen cells is always at least equal and sometimes substantially greater than that observed for cells seeded from isolation.

With regard to thymidine, for the experiments conducted, the results show a greater variability in the level of incorporation of the labeled precursor: this incorporation may be equivalent and slightly greater or less than that of the non-frozen control cells, without the values deviate significantly. Moreover, there is no significant difference between the results obtained after various contact times with the
DMSO.

- NEOSYNTHESIS OF PROTEINS BY ELECTROPHORESIS
AND AUTORADIOGRAPHY OF ELECTROPHORESIS
The electrophoresis and autoradiographs of electrophoresis prove that the frozen cells defrosted, after 7 days of culture, neosynthesize proteins and that these proteins are the same as those revealed in the non-frozen control cells.

The results are shown in Figure 27.

Lanes 1, 2, 3, 4, 5, 6 correspond to 1 - Non-frozen control cells - 1st assay 2 - Frozen-thawed cells 3 - Unfrozen control cells - 2nd assay 4 - Frozen-thawed cells 5 - Nonfrozen control cells - 3rd test 6 - Frozen-thawed cells
The protein profile for frozen cells is similar to that obtained for cells seeded in isolation. The predominantly neosynthesized proteins are observed in both cases.

- NEOSYNTHESIS OF LIPIDS BY CHROMATOGRAPHY
ON COUCHEMINCE AND AUTORADIOGRAPHY OF CHROMATOGRAM
Experiments carried out to verify that the cells after freeze-thawing neosynthesized the same types of lipids as the cells "freshly" isolated, allow to observe by autoradiography of thin layer chromatograms (FIGS. 28A and 28B) that these compounds, neosynthesized after 7 days of culture by the cells frozen from 14C acetate, are the same as those synthesized by the cells seeded from isolation.

Figure 28 A corresponds to neutral lipids, and Figure 28B to polar lipids and lanes 1, 2, 3 1 - to cells seeded after dissociation 2 - to cells seeded after freezing
Thawing (10 min contact with DMSO) 3 - To cells seeded after freezing
thawing (15 min contact with DMSO).

 In fact, whether for neutral or polar lipids, the lipid profiles are identical. In contrast, for polar lipids, quantitative differences are noted as evidenced by the intensity of the spots. The same observation is made with regard to neutral lipids. Frozen-thawed cells appear to synthesize lipids in greater amounts than the same cells seeded after isolation.

 In addition, 10 minutes or 20 minutes of contact with DMSO before freezing of the cells does not alter the profile of neosynthesized lipids.

III APPLICATIONS AS MODELS IN VITRO EN
TOXICOLOGY
Suspended cells (scallop gill cells and oyster embryo cells) or cultured (scallop heart cells) were used as bioassays to evaluate the cytotoxicity of heavy metals and pesticides and the genotoxic effect of a reference mutagenic substance.

 The cytotoxicity tests selected for the reported tests are the measurement of the oxygen consumption of the cells and the FDA test.

Measuring oxygen consumption
The effect of various heavy metals on the respiration of dissociated cells was evaluated by measuring the oxygen consumption of oyster embryonic cells and scallop gill cells suspended in seawater.

 The oximeter (Gilson) is first calibrated; the baseline (100% oxygen) is set on bubbled oxygenated seawater introduced into the measuring vessel with a Clark electrode. A recorder using a thermosensitive graph paper makes it possible to control the stability of this "100% oxygen" line.

Zero is adjusted by addition of sodium dithionate.

Once the calibration has been carried out, the tank is thoroughly rinsed with seawater. The cells are then introduced into the thermostatically controlled measuring tank at 150 ° C. and the oxygen consumption is monitored for a few minutes, until the slope is constant.

 The heavy metals to be tested are then added to the cell suspension and their effect on oxygen consumption is measured. A slope failure caused by the addition of metals, i.e. the decrease in oxygen consumption of the cells per unit of time (given by the speed of unwinding of the paper), reflects the toxicity of the products. Only high doses of metals, mercury chloride (HgC12), zinc chloride (ZnCl2), copper chloride (CuCl2) were tested (10-3 to 10-5 M) in order to optimize the technique.

FDA test
The high sensitivity fluorescein diacetate (FDA) deesterification test was used to assess the cytotoxicity of heavy metals (copper, lead, zinc, cadmium) on cultured scallop core cells.

 After enumeration, the heart cells are cultured in 96-well plates, at a rate of 250,000 viable cells per 100 μl of culture medium and per well. The culture medium is renewed every three days and the cultures are kept in incubation for eight days. Many cardiac cells adhere to the culture medium after this time.

They are then put in contact with the toxic for 18 hours.

The FDA test was carried out following the protocol of Minier et al. (1993). After removing the culture medium, the cells (four wells per dose of toxic, four control wells) are incubated in the presence of FDA. The FDA (Sigma) solubilized at 0.333 mg / l in acetone is dissolved in seawater to obtain a final concentration of 40 μM. 100 μl of this solution is added to each well. A fluorescence measurement is carried out after eight hours. The readings are made with the fluorimeter (Fluorolite 1000,
Dynatech) equipped with 485 nm filters for excitation and 538 nm for emission. Fluorescence measurements are arbitrarily expressed in fluorescence units.

 The measurement of oxygen consumption makes it possible to demonstrate the acute toxicity of certain heavy metals, at certain concentrations, with respect to scallop and oyster cells. Thus, the addition of mercury chloride to gill cells in suspension decreases the oxygen consumption of the cells, which is reflected in the recordings by a break in slope. This inhibitory effect of metal on cellular respiration is observed for final concentrations ranging from 10-2M to 10M and is dose-dependent. FIG. 29 shows the oxygen consumption of these cells for a mercury chloride concentration of 10 -2 M. A concentration of 10 3 M of mercury has the same inhibitory action on the oxygen consumption of the embryonic cells of the cells. 'Oyster.

 At a final concentration of 10-3M, zinc chloride and copper have the same inhibitory effect as mercury.

 Figure 30 illustrates these results compared to control plots obtained for cells of the same batch, in the absence of toxic on the one hand and in the presence of potassium cyanide, known inhibitor of breathing on the other hand.

 The addition of FDA to the cultured cells makes it possible to obtain a response whose intensity is dependent on the concentration of substrate. This hydrolysis of the FDA grows rapidly as a function of the incubation time of the cells with the product for the doses of 10, 16, 20, 32 and 40 μM. For 32 and 40 μM substrate, a plateau phase is obtained between 8 and 10 hours.

 Measurements after 8 hours of minimum contact with 40 μM of FDA were performed so that the concentration of substrate is non-limiting and meet both the objectives of standardization and rapidity of the test.

 The results obtained for the FDA test, applied to primary cultures of stellate shell heart cells after seven days of culture and 18 hours of contact only with heavy metal salts (lead, copper, cadmium, zinc) compared to control cultures, are illustrated in FIGS. 31A to 31D. The statistical analysis of the data was made in this case by the Mann-Whitney U-test.

 A spontaneous hydrolysis control of fluorescein diacetate made it possible to verify that this phenomenon remains of low intensity (of the order of 10%) and that the fluorescence measured during the tests is attributable essentially to the cells cultured.

 This study of the impact of "heavy metals" on the esterase activity of cells shows that 18 hours of incubation in the presence of lead, even at a dose of 103M, do not cause a significant variation in FDA hydrolysis. In contrast, inhibition is observed for the highest concentrations of copper, zinc, and cadmium. A zinc concentration of 10M appears cytotoxic. The esterase activity at 8 hours is significantly different from that of the control cultures. In the case of cadmium and copper, the concentrations 102M and 103M strongly inhibit the hydrolysis of FDA by the heart cells.

 In addition, for these last doses, the metal ions cause the detachment of the adherent cells in culture.

 The invention thus provides a protocol for isolating cells. It is a set of conditions for carrying out the culture of marine invertebrate cells and means for adapting to analytical cells of marine origin analytical methods commonly used in cell culture.

 The provisions defined above make it possible routinely to carry out, without any problem of microbial contaminations, primary cultures of marine invertebrate cells, with the maintenance in vitro of major activities of biosynthesis, as illustrated above with the bivalve cells.

 The invention also provides the means for obtaining cells and biological material in the frozen - thawed state of marine Invertebrates whose biosynthetic activity is preserved as demonstrated by comparison with controls. Such cultures and biological materials offer many advantages in terms of ease of process realization, non-dependence on the natural resource and standardization for applications in biotests, particularly in vitro toxicology or pathology. .

Bibliographical references
BLIGH EG & DYER WJ (1959). A rapid method of total lipids extraction and purification. Can. J.

Biochem. Physiol., 37: 912-917.

 BRADFORD M. (1976). A rapid and sensitive method for the quantification of micrograms of protein using the principle of protein-dye binding.

Anal. Vbiochem., 72: 248-254.

 GRUFFYDD L.D. & BEAUMONT A.R. (1970).

Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximux (Mollusca, Lamellibranchia). Helgol Wiss.

Meeres., 20: 486-497.

 LABARCA C. & PAIGEN K. (1980). A simple, rapid and sensitive DNA assay procedure. Anal. Biol., 102,344-352.

LAEMMLI UK (1970). Cleavage of structural proteins during the assembly of the bacteriophage
T4. Nature, 227: 680-685.

LANITZI. (1986). Autoradiography. Animal Cell
Culture. Ed. RI Freshney. Irl. Press., 167-179.

 METCALFE L.D. & SCHMITZ A.A. (1951). The rapid preparation of fatty acid esters for gas chromatographic analysis. Analytical Chemistry, 33: 363-364.

 OSHIMA S.L., RATNAYAKE W.M.N. & ACKMAN R.G.

(1987). Cod lipids, solvent systems and the effect of fatty acid lenght and unsaturation on lipid class analysis by Iatroscan TLC-FID. J. AOCS., 64: 219-223.

 RATNAYAKE W.M.N. & ACKAN R.G. (1985). Rapid analysis of canola lipid composition by Iastrocan

Claims (21)

 1. A method for culturing marine invertebrate cells, characterized in that it comprises the incubation of isolated cells, viable, substantially free of contaminants, at a temperature and in a medium whose composition, based on sterile seawater, and the physicochemical properties, in particular osmolarity and pH, are chosen so as to obtain viable cells and maintain or improve their functional capacities for at least 30 days. 2. A process according to claim 1, characterized in that the isolated cells cultured are as obtained from explants or by dissociation of tissues and after a decontamination pretreatment. 3. A process according to claim 2, characterized in that the dissociation of the tissues is carried out using a chemical system and / or an enzymatic system and / or a mechanical system. 4. A process according to claim 3, characterized in that the dissociation of the tissues is carried out using pronase, preferably at concentrations of 0.020 to 0.030%, in particular of 0.025% n for about 12 hours, at a temperature of not exceeding 40C. 5. Method according to one of claims 1 to 4, characterized in that the decontamination of tissues is carried out by treatment with antibiotics and, preferably, mucolytic agent, in particular with the aid of antibiotic solution comprising penicillin, streptomycin, kanamycin, gentamycin, alone or in admixture, and a mucolytic agent consisting of dithiothreitol. 6. A process according to one of claims 1 to 5, characterized in that it comprises a step of pretreatment animals with the use of brushing and / or the action of disinfectants and / or handling in a sterile atmosphere and or washing with large volumes of sterile seawater and / or dissecting tissues under sterile conditions and / or treating the tissues with antibiotic and / or mucolytic baths. 7. A process according to claim 6, characterized in that the animals collected are housed in hatcheries, in seawater, for example for a fortnight, then left to rest in tanks fed with recycled seawater and sterilized by Uv. 8. A process according to one of claims 1 to 7, characterized in that the incubation step is carried out with viable cells seeded at a rate of 1 to 2 million cells per ml of medium, in particular of the order of 1.5 million / ml. 9. A process according to one of claims 1 to 8, characterized in that the osmolarity of the culture medium is of the order of 800 to 1300 mOsm, preferably of the order of 1100 mOsm, and the pH of 7. , About 3 to 8. 10.- Method according to one of claims 1 to 9, characterized in that the sterile seawater-based culture medium is buffered and supplemented with additives such as present in the Leibovitz medium, in particular the medium of Leibovitz-15 at about 10 to 208 (V / V), in particular about 15. 11. A process according to claim 10, characterized in that the culture medium contains serum, in particular decomplemented serum, in a proportion of 5 to 15% (V / V), in particular approximately 10%. 12. The method of claim 10 or 11, characterized in that the culture medium comprises lipids, especially unsaturated lipids of marine origin. 13.- Method according to one of claims 8 to 12, characterized in that the incubation step is carried out at a temperature of the order of 15 to 16 ° C, in a normal atmosphere not enriched in CO2. 14. Method according to one of claims 1 to 13, characterized in that the cells used are embryonic cells or differentiated cells of haemocytes and various organs such as, depending on the species, coat, palpi labials, muscles, gills, digestive gland and heart. 15.- Method according to one of claims 1 to 14, characterized in that the cells come from Molluscs, in particular of bivalves such as ostreids, mytilids and pectinids, Gastropods and Cephalopods, or Echinoderms such as Astenidae and Echinids, or Arthropods, especially Crustaceans such as shrimps. 16.- Method for freezing / thawing a biological material comprising isolated cells as used in the method according to one of claims 1 to 7, 14 or 15, cells collected after culture according to the method of the one of claims 1 to 15, and the tissues and organs from which these cells are derived, characterized in that it comprises contacting said biological material, in a culture medium as defined above, with an agent cryoprotectant, such as dimethylsulfoxide, then slow freezing at 1 to 50 min at a temperature below -200C, preferably below -500C, preferably of the order of -80 C and immersion in liquid nitrogen. 17.- Method according to claim 16, characterized in that, to thaw the biological material, the containers containing the container at a temperature above ambient, preferably from 30 to 400C, in particular 37 " C, then the freezing medium is diluted with nutrient culture medium, the cells are separated, for example by centrifugation, and resuspended for seeding. 18. Cell cultures and biological material as obtained by the method according to one of claims 1 to 17. 19. Cell cultures according to claim 18, characterized in that they consist essentially of viable cells, capable of neosynthesizing DNA, proteins and lipids, in particular unsaturated lipids. 20. Application of cell cultures and biological material according to claim 18 or 19 as models in in vitro bioassays, in particular in toxicology and pathology. 21. Kits for the implementation of bioassays, characterized in that they comprise a biological material or a cell culture according to claim 18 or 19 with the appropriate appropriate culture media and reagents for carrying out the bioassays.