WO1993011224A1 - Culture media for cells of marine invertebrates - Google Patents

Abstract

A supplement is disclosed for cell culture media suitable for the culture of marine invertebrates, particularly molluscs. The composition comprises at least one hormone selected from the group comprising insulin, IGF (Insulin Like Growth Factors), hydrocortisone and glucocorticoids, at least one growth factor selected from the group comprising EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), and PDGF (Platelet Derived Growth Factor), at least one substance or a lipid extract selected from the group comprising Ex-Cyte VLE, egg yolk extract, and prostaglandin F2$g(a), and at least one detoxifiant selected from the free radical inhibitors. Culture media containing the said supplement, and a method for the culture of marine invertebrate cells in the presence of said supplement and in an oxygen-lean atmosphere are also disclosed.

Description

 CULTURE MEDIA FOR MARINE INVERTEBRATE CELLS.

The invention relates to new cell culture media suitable for cell culture of marine invertebrates, in particular for cell culture of molluscs.

Invertebrate cell cultures first appeared in the 1930s. They were mainly oriented towards insect cell cultures, due to their numerous applications in virology and pathology. These cultures have since grown rapidly and are now used on an industrial scale for the production of recombinant proteins (baculovirus system). It is not the same for other inver¬ tebrates, and particularly for molluscs; attempts to culture mollusc cells have been quite numerous, but punctual and little followed; few species have been cultured, and the work undertaken has not resulted in the development of a culture medium allowing significant cell proliferation in vitro.

The first culture media for marine mollusc cells consisted of filtered, buffered seawater, supplemented with antibiotics, carbohydrates and nitrogen in the form of amino acids or protein extract [VAGO and CHASTANG , CR Acad. Sci. , 250, 2751-2753 (i960)].

Then analytical knowledge on the composition of the hemolymph (the extracellular fluid bathing the cells of invertebrates) was acquired, and the composition of the culture media was modeled on the composition of the hemolymph: the culture medium is the reconstitution as faithful as possible to the organic and mineral composition and the physicochemical characteristics of the hemolymph of the animal placed in culture [CHERNIN, J. Parasitol., 49, 353-364 (1963)].

However, the composition of the hemolymph varies between different groups of invertebrates, and within the same genus, between species. This composition also has great intraspecific variability: the composition of the oyster hemolymph varies, for example, depending on the season and the habitat.

It is therefore necessary, prior to any cultivation, to study beforehand and very completely the composition of the hemolymph of each species cultivated, which implies very complex and very heavy preliminary analytical work. In addition, the environments developed by this method must be modified with each change of cultivated species. An effort to standardize culture media for invertebrate cells was undertaken in 1969 by VAGO and QUIOT [Ann. Zool. School. Anim. , 1, 281-288 (1969)].

They established different formulations, based on the Na / K ratio, the osmotic pressure and the pH of the hemolymph of the cultivated species. The culture media thus developed break down as follows:

- a constant part comprising an organic fraction (carbohydrates, lipids and amino acids), antibiotics, and serums providing vitamins and non-specific growth factors.

- a part which varies according to the species cultivated, comprising a mineral fraction (mineral salts in specific proportions, making it possible to adjust the osmotic pressure to the desired value), a special fraction (specific vitamins and growth factors) , and a fraction allowing the pH to be buffered to the desired value.

The physico-chemical characteristics of the environments respect those of the hemolymph of cultivated species: for marine molluscs, the cells are generally cultivated between 15 "and 25 ^* C, at a pH varying between 6.8-7.0 and 7.2-7.4 and at the same osmotic pressure as that of the hemolymph, ie between 900 and 1100 mosmol / 1. The culture supplements used are very varied: so-called "indefinite" supplements, the chemical composition of which is not specified, such as the homologous or heterologous hemolymph inactivated, at 5 or 10%, fetal calf serum at 10%, bovine serum, horse serum, extracts of organs or whole animals, embryonic chicken extracts, or so-called "defined" supplements such as: protein hydrolysates, amino acids, vitamins, factors specific or non-specific growth, etc. Although the supplements mentioned above are commonly used for vertebrate cell cultures, their use for the culture of marine invertebrate cells has so far been unsuccessful to satisfactory results. This is explained by the fact that the cells of marine invertebrates live in natural conditions very different from those of vertebrates. Marine invertebrates and more particularly bivalve molluscs do not have an interior environment proper, and are not capable of homeostasis. They have specific enzyme systems, adapted to lower temperatures (15- 20 ^° C) and at higher pressures; the substrates do not have the same affinities for these enzymes as for vertebrate enzymes. (Km, etc ...). On the other hand, to reproduce as much as possible the composition of the hemolymph, the cultures are carried out in hyperosmotic environments: (1100 mosmol / 1, or 3 to 4 times more than the conventional osmolarity of the environments for vertebrates which is about 300 mosmol / 1). In order to achieve this osmolarity, the salinity of the media is brought to about 35 g / l of dissolved salts. However, it is known that hypersaline conditions, the permeability of the membranes is impaired, and the exchange capacities between the cells and their environment reduced.

The satisfactory culture conditions for vertebrate cells cannot therefore be transposed a priori to the culture of marine invertebrate cells.

Certain environments, more specifically responding to the needs of certain types of marine invertebrate cells, have been developed; PCT Application US 88/03325 describes, for example, a culture process allowing cells of explants from oyster coats intended for pearl production to be kept alive, in a medium comprising a saline solution simulating the natural medium associated with the tissue oyster coat; amino acids, assimilable sugars, and a supplement such as hemolymph, mollusc gonad extracts, yeast extract or fetal calf serum. The use of such media allows maintenance in primary cultures of adult cells, therefore differentiated, of molluscs, for periods ranging from a few days to several months. However, these cultures do not show mitotic figures, or very few and only during the early days of culture. The mitotic cell index is very low: 1 "/ ^* 'in white body cell cultures of Octopus vulgaris and 1 to 5 ^* /"' in somatic cell cultures of Marine Bivalves. Primary cultures from explants contain cells in suspension and adherent cells, which form a confluent carpet after 7 to 15 days of culture. From suspended cells, adherent cells, or from all of the primary culture cells, attempts have been made to obtain secondary cultures. However, secondary crops do not form no carpet converges and therefore cannot be transplanted again; they can only be kept alive for periods ranging from a few days to several weeks in vitro, without proliferating significantly; their maintenance depends on the culture conditions (presence / absence of explants, seeding density, ...) and the characteristics of the culture media used.

Unlike adult cells, the cells of Crassostrea gigas and Crassostrea virginica larvae appear to divide actively in primary culture. Biomphalaria glabrata larva cells actively divide and can be transplanted for several generations. However, their culture was always carried out over short periods, following contaminations which appeared and developed rapidly at the expense of the cultured cells.

The present invention, which was produced by the Applicant in collaboration with the Laboratory of Marine Invertebrate Biology of the Museum of Natural History, has set itself the goal of providing culture media which respond better than the media of ancient art. ¬ laughing at the needs of marine invertebrate cells, in particular molluscs, and allowing the culture of adult cells, ensuring longer survival, and better proliferation of these cells in culture and in particular in secondary cultures.

For this, the inventor has developed a culture medium which comprises, in addition to a basic mineral and organic medium, a new supplement, hereinafter called "defined supplement", and possibly, a so-called "indefinite" supplement (c (i.e. a serum or hemolymph supplement, the exact chemical composition of which cannot be determined with precision). The constituents of the defined supplement according to the invention have already been described, among others, as active on the growth of cells in culture, in the case of cells of mammals and other vertebrates. However, the association of these constituents had never been proposed before, nor had it been suggested that they could have a favorable effect on the viability of the cultures of cells of marine invertebrates, and in particular of molluscs.

The subject of the present invention is a composition which can be used as a defined supplement for a cell culture medium intended for the culture of marine invertebrates, which composition is characterized in that it comprises, at least one hormone chosen from the group comprising insulin, IGF (Insulin like Growth Factor), hydrocortisone and glycocorticoids, at least one growth factor chosen from the group comprising EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), and PDGF (Platelet Derived Growth Factor), at least one lipid substance or extract chosen from the group comprising Ex-Cyte VLE, egg yolk extract, and prostaglandin F2α, and at least one protective agent chosen from free radical inhibitors.

According to a preferred embodiment of the supplement defined for cell cultures in accordance with the invention, it comprises between 10 and 100 μg / ml of bovine insulin, between 10 ^~ ^ and 10 ^{~ 4} M hydrocortisone, between 5 and 50 ng / ml of EGF, between 1 and 25 ng / ml of PDGF, between 0.2 and 2% (v / v) of egg yolk extract, between 0.05 and 0.2% v / v of Ex-cyte VLE, between 10 ^{~ 9} and 2 x 10 ^{~ 8} M prostaglandin F2α, between 10 and 100 μg / ml of catalase.

The inventor further noted that the effect of the supplement defined in accordance with the invention was considerably increased when the said supplement was combined with a supplement of the "indefinite" type such as fetal calf serum or hemolymph. oyster. The invention also encompasses cell culture media suitable for the culture of marine invertebrate cells, in particular molluscs, which media are characterized in that they comprise a basic medium comprising mineral salts, sugars, acids amino, and vitamins, to which is added a defined supplement for cell cultures in accordance with the invention.

According to a preferred embodiment of the cell culture media in accordance with the invention, they also comprise an indefinite supplement chosen from the group consisting of fetal calf serum or oyster hemolymph.

The media in accordance with the invention consist for example of a commercial mineral and organic base (RPMI 1640, LEIBOVITZ L-15, etc.) to which mineral salts are added (NaCl, KC1, CaCl2, MgCl ₂ , MgSθ4), antibiotics (penicillin, streptomycin) and supplements of indefinite composition (fetal calf serum, hemolymph, ...) and a supplement of defined composition in accordance with the invention.

The present invention further relates to a method of culturing marine invertebrate cells, characterized in that said cells are cultured in a medium comprising the defined supplement according to the invention.

The inventor also found that the development and survival of cultures of marine invertebrate cells are further improved when the use of culture media comprising the defined supplement in accordance with the invention is combined with the culture of cells in an atmosphere. poor in oxygen.

According to a preferred embodiment of the method of culturing marine invertebrate cells according to the invention, said cells are cultured under an atmosphere containing less than 10% oxygen.

According to an advantageous modality of this mode of implementation, the cells are cultured under an atmosphere containing less than 5% oxygen. The defined supplement in accordance with the present invention has the following advantages over supplements of the prior art: it improves the short-term viability and the long-term survival of cultures of marine invertebrate cells; it can replace fetal calf serum in these cultures, and acts in synergy with it to improve cell viability and survival; its effect is increased by the reduction of the partial oxygen pressure. The present invention will be better understood with the aid of the additional description which follows, which refers to examples of culturing mollusc cells in a medium in accordance with the invention.

It goes without saying, however, that these examples are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

The experiments described below were carried out on cultures of hollow oyster Crassostrea gigas heart cells.

Example 1: Preparation of the culture media The basic medium used is a commercial medium, for example LEIBOVITZ L-15 medium (SEROMED), enriched with mineral salts. The composition of a complete basic medium which can be used is indicated by way of example in Table I below:

TABLEAU I

TABLE I

TABLEAU I (suite)

TABLE I (continued)

AMINO ACIDS g / i

L-Alanine 0.225 L-Arginine 0.5 L-Aspargine 0.25 L-Cysteine 0.12 Glycine 0.2 L-Histidine 0.25 L-Isoleucine 0.125 L-Leucine 0.125 L-Lysine 0.075 L-Methionine 0.075 L- Phenylalanine 0.125 L-Serine 0.2 L-Threonine 0.3 L-Tryptophan 0.02 L-Tyrosine 0.3 L-Valine 0.1 L-Glutamine 0.294 (at time of use)

To this medium, a defined supplement in accordance with the invention is added in the form of a concentrated FPL stock solution, prepared in advance, distributed in aliquots and stored at −20 ° C.

La composition de cette solution stock est indiquée dans le tableau II suivant :

The composition of this stock solution is indicated in Table II below:

TABLE II

In addition, any of the following undefined supplements can be added to the middle:

- Fetal Calf Serum (SVF) [PAA GmbH] decomplemented by heating for 30 'at 56 ^* C, or

- Homologous hemolymph [hemolymph of hollow oyster (C. gigas) taken by puncture of the pericardial cavity of oysters during winter, (October-March), filtered through 0.2 μm and frozen at -20 ° C], inactivated by heating for 30 'at 56 ^* C).

The pH of the media is adjusted to 7.0-7.5, their osmotic pressure at 1000-1100 mosmol / 1 and the cultures are incubated at 18-25 ° C in air: 80% N ₂ , 20% 0 ₂ , 0 , 03% C0 ₂ (v / v). Example 2 Effect of the FPL Supplement on the Viability of the Cells in Culture

- Cell viability test

The cell viability test chosen is the assay for the reduction of MTT ((-3- (4,5-dimethylthiazol-2-yl) -2,5 diphenyl tetrazolium bromide) by the mitochondrial succinate dehydrogenases of living cells.

This test is carried out according to the following protocol:

The cells are adjusted to successive dilutions in the culture medium and seeded in 96-well microplates: 100 μl of cell suspension / well, 3 repetitions / dilution; 10 μl of a solution of MTT at 5 mg / ml in PBS are added to each well; the microplate is incubated for 6 h at 20 ° C; 100 μl of 0.04 N HCl in isopropanol are added to each well; the plate is stirred for 10 minutes; the OD (optical density) is read at λ = 570 n against λ ref. = 630 min. Only living cells can reduce MTT to formazan. At a given instant and in a given physiological state, the quantity of formazan formed (OD 570 n / OD 630 nm) is directly proportional to the number of viable cells in culture. This linearity is respected over 24 h for a range of 0.1 to 1.5 × 10 ° viable cells / ml.

The ability of cells to reduce MTT (dDO / d number of viable cells) is specific to a given cell population, in a given physiological state. This capacity varies during the cultures.

This test therefore provides an overall indication of the number, activity and viability of the cells in culture; it makes it possible to distinguish between death, survival and proliferation of cells; it makes it possible to measure cell activation, even in the absence of proliferation. - Study of the effect of the FPL supplement

The heart cells of C. gigas oyster were cultivated, in primary cultures from explants, or in secondary cultures, in different media, respectively: a) basic medium; b) basic medium supplemented with 10% v / v of undefined supplement (fetal calf serum); c) base medium supplemented with 10% v / v of stock solution of defined supplement FPL; d) base medium supplemented with 10% v / v of stock solution of defined supplement FPL, and 10% v / v of undefined supplement (fetal calf serum).

The behavior of cells in these different media has been determined:

- on cells in primary culture, by observing cell morphology, evaluating migration and total cell yield, and evaluating cell viability and adhesion.

- on cells in secondary culture, by measuring the capacity of cells to reduce MTT (MTT test performed according to the protocol described above), as well as by observing cell morphology, assessing density , sustainability and

Cell adhesion.

The results are as follows:

1) Ability of cells to reduce MTT:

(short-term test, over 7 days) - In the presence of 10% FPL supplement, the cells' capacity to reduce MTT increases from 25 to

30% compared to that observed with the base medium; in the presence of 10% fetal calf serum, the response is identical. - In the presence of 10% FPL supplement + 10% fetal calf serum, the ability of cells to reduce MTT increases by 100%.

2) Survival of secondary crops (long-term test, over 3 months)

- In the presence of 10% FPL supplement + 10% fetal calf serum, about 40% of surviving cells remain after 15 days of culture, while less than 30% remain when the cultures are carried out in the basic medium without supplement; in addition, during the second month of culture, an increase of 35 to 50% in the number of viable cells is observed in the presence of 10% of FPL supplement + 10% of fetal calf serum, while in the presence of 10% of fetal calf serum alone, the number of viable cells remains stable.

The defined supplement in accordance with the invention increases the mitochondrial activity of cells in culture in vitro, and also acts in synergy with other ^" supplements such as fetal calf serum. Metabolic activation is also accompanied by particularly significant cell proliferation during the second month of culture It therefore appears that the defined supplement in accordance with the invention makes it possible to improve the viability and proliferation capacities of adult cell cultures differentiated from marine invertebrates.

Example 3 Effect of Reducing the Partial Oxygen Pressure on Secondary Culture Cells in the Presence or Absence of the FPL Supplement

Secondary cultures were carried out by trypsinization and subculturing of adherent cells coming from primary cultures in explants aged either 7 days (1st experiment), or 14 days (2nd experiment). The subcultured cells were seeded in secondary culture in T-25 CORNING flasks at 40-45,000 cells / cm ² , in 2 ml of medium supplemented by the combination of 10% FCS and 10% FPL. The cultures were incubated at 20 ° C., either under 20% of 0 ₂ , or under 2% of 0 ₂ . Their evolution was followed over time, by morphological observation, and by enumeration of all cells on the Malassez slide and trypan test: cell types present, total number of viable cells excluding trypan blue, and cell adhesion. The first experiment was followed over 43 days. The second experiment was followed over 28 days.

The secondary cultures produced at 20% and 2% 0 contain the same cell types: amoebocyte type cells, epitheliocyte type cells and fibroblastic (contractile) cells.

The first experience highlights a difference ^'survival as the cultures were incubated with 2 or 20% 0 _2: during the first two weeks of culture, the viable cell density decreases in both 20% 0 ₂ and under 2% of 0: around 60% of surviving cells remain after 14 days; then the density of viable cells increases, slightly (+ 25%) and from 28 days in cultures incubated with 20% O ₂ , more strongly (+ 50%) and from the 14th day on in cultures incubated with 2 % of 0; after 43 days, there remains under 2% of O ₂ about 90% of surviving cells, while under 20% of O ₂ there remains only about 60%.

The second experiment confirms this difference in survival observed between secondary cultures incubated under 20% O ₂ and under 2% O ₂ : after 28 days, there remains under 2% O ₂ approximately 50% of cells. survivors, whereas under 20% of 0 ₂ , only about 40% remain. The reduction of the partial pressure in O ₂ therefore improves the survival of the secondary cultures of oyster heart cells: it accelerates and amplifies the phenomenon of transient growth observed during the second month of culture in medium supplemented by the association 10% of SVF and 10% FPL.

A third experiment of secondary culture under 20% or under 2% of O ₂ was carried out, in medium not containing FPL, in order to see if the response of the oyster cells to the decrease in the partial pressure in 0 ₂ depends on the composition of the medium in which they were cultivated, and in particular on the presence of the FPL supplement.

Secondary cultures were carried out by trypsinization and subculturing of adherent cells from primary cultures in explants aged 14 days. The subcultured cells were seeded in secondary culture in T-25 CORNING flasks at 65,000 cells / cm ² , in 2 ml of medium supplemented with FCS but not supplemented with FPL. The cultures were incubated at 20 ° C., either under 20% of 0, or under 2% of 0 ₂ .

Their evolution was followed over time, by morphological observation and by counting all cells on the Malassez slide and trypan test: cell types present, total number of viable cells excluding trypan blue and cell adhesion. This experiment was followed over 43 days.

In the absence of the FPL supplement, the evolution of the cultures incubated under 20% or under 2% of O is identical: there is no transient growth; the density of viable cultures decreases and then stabilizes at around 40% of surviving cells after 28 and 43 days of culture.

In the absence of an FPL supplement, the reduction of the partial pressure to 0 therefore has no effect on the proliferation of secondary cultures. In conclusion, reducing the oxygen pressure in the atmosphere improves the development and survival of oyster heart cell cultures: in primary culture by explants it slows cell migration and prolongs the maintenance of epitheliocyte cell types and fibroblastic, without changing the yield after two months; in secondary culture it accelerates and amplifies the transitory growth phenomenon observed during the second month of culture in medium supplemented with 10% of FCS and 10% of FPL; this positive influence is observed in the presence of the FPL culture supplement, but not in the presence of fetal calf serum alone.

Claims

1) Composition which can be used as a supplement for a cell culture medium suitable for the culture of marine invertebrates, in particular molluscs, which composition is characterized in that it comprises, at least one hormone chosen from the group comprising insulin, IGF (Insulin like Growth Factors), hydrocortisone and glucocorcoids, at least one growth factor chosen from the group comprising EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor) and PDGF (Platelet Derived Growth Factor), at least one lipid substance or extract chosen from the group comprising Ex-Cyte VLE, egg yolk extract, and prostaglandin F2α, and at least one detoxifier chosen from free radical inhibitors. 2) Supplement for cell cultures according to Claim 1, characterized in that it comprises between 10 and 100 μg / ml of bovine insulin, between 10 ^{~ 6} and 10 ^{~ 4} M of hydrocortisone, between 5 and 50 ng / ml EGF, between 1 and 25 ng / ml of PDGF, between 0.2 and 2% (v / v) of egg yolk extract, between 0.05 and 0.2% of ex-cyte VLE, between 10 ^"9 and 2 x 10 ^{" 8} M of prostaglandin F2α, between 10 and 100 μg / ml of catalase. 3) Cell culture medium suitable for the culture of marine invertebrate cells, characterized in that they comprise a basic medium comprising mineral salts, sugars, amino acids, vitamins, to which is added a supplement for cell cultures according to Claim 1. 4) Cell culture medium according to Claim 3, characterized in that it further comprises a supplement chosen from the group consisting of fetal calf serum or oyster hemolymph. 5) Process for culturing marine invertebrate cells, characterized in that said cells are cultured in a medium according to any of claims 3 or 4. 6) A method of culturing marine invertebrate cells according to claim 5, characterized in that the cells are cultured under an atmosphere containing less than 10% of oxygen. 7) A method of culturing marine invertebrate cells according to claim 6, characterized in that the cells are cultured under an atmosphere containing less than 5% oxygen.