Cytotechnology 16: 109-120, 1994. 109 @ 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Identification of media supplements that improve the viability of primarily cell cultures of Crassostrea gigas oysters

Isabelle Domart-Coulon, Dominique Doumenc, Stephanie Auzoux-Bordenave and Yann Le Fichant t Laboratoire de Biologie des Invertgbr~s Marins CNRS URA 699, Museum National d'Histoire Naturelle, 57 rue Cuvier 75005 Paris, France; I Bertin & Cie, B.P 3, 78373 Plaisir Cedex, France

Received 2 August, 1994; accepted 2 August, 1994

Key words: Cell culture, media development, MTT, Bivalvia, oyster

Abstract

Media supplements have been investigated for their influence on the viability of primary cell cultures from the heart of Crassostrea gigas oysters. Soluble factors of vertebrate origin were tested, belonging to five families of supplements that had proven to increase the viability of insect and mammal cell cultures. Using two-level complete factorial assays, factors and mutual interactions were screened within each family with a MTT reduction assay. Results pointed out the positive influence of hormones, growth factor, antioxidants and lipids on the mitochondrial metabolism of oyter's heart cells. Consequently, a new concentrated complex supplement was developed. At 10% (v/v) final concentration in modified Leibovitz L-15 medium, it increases by 30% the cellular viability of one-week old cultures as compared with non-supplemented medium, a similar improvement as the one obtained with 10% (v/v) fetal calf serum. Combined with fetal calf serum, this new supplement doubles the cellular viability of one- week old cultures and allows networks of cardiomuscular cells to be maintained functional over three months in vitro.

Abbreviations: MTT-3-(4,5-dimethylthiazol;-2-yl)2,5-diphenyl-tetrazolium bromide; FCS-fetal calf serum

Introduction

Viable cell cultures derived from marine inverte- brates are needed for numerous promising applica- tions: pathology diagnosis, studies on disease treat- ment, growth and reproduction control of commercial shellfishes, design of new toxicological tests for mon- itoring water quality; such cultures could also open the way to the in vitro production of complex marine molecules with proven pharmacological or industrial interest.

For all these reasons, the in vitro maintenance and propagation of cells of marine invertebrates has been attempted since the early 1960's (Vago and Chastang, 1960). Either embryos, larvae or adults are used, most- ly from bivalves and crustaceans because of their com- mercial interest but also from cephalopods, echino- derms, cnidarians and sponges. These cultures are not

yet as successful as cultures from mammalian, fish or insects: primary cultures of marine invertebrate cells are maintained viable for time intervals ranging from several hours (Lenoir, 1989) to several weeks (Auzoux et al., 1993) or months (Odintsova and Khomenko, 1991; Klautau et al., 1993) but their functionality is uncertain and their growth is limited; although a few authors report increases in ceil numbers (Brewster and Nicholson, 1979; Klautau et al., 1993) or mitotic figues (Odintsova and Khomenko, 1991), attempts to serial- ly propagate these cells have failed and continuous cell lines have not yet been established; best results were obtained with embryonic or larval tissues (Ellis and Bishop, 1989) and with spongesPolymastia which cells have recently been claimed to have been main- tained replicating for more than two years (Klautau et al., 1993).

110

A major difficulty is the frequent contamination with protozoa, bacteria and fungi that causes loss of most cultures. Most authors also point out the lack of data on the in vitro physiology of the cells and the resulting absence of appropriate culture media and conditions to explain the low frequency of mitosis observed (Mialhe et al., 1988). Indeed, up to now, no extensive and systematic study of media compo- nents and culture conditions has been reported. Cells are maintained in nutritive culture media to which sup- plements are added. Except for a few trials with natural or artificial seawater (Vago and Chastang, 1964; Cecil, 1969; Klautau et al., 1993), most culture media consist of balanced saline solutions complemented with car- bohydrates and amino-acids; Antibiotics are frequently added in order to prevent microbial contamination. A first strategy is to make media de novo, the aim being to accurately mimic the composition of hemolymph of the bivalve or the crustacean that is put into culture (Machii and Wada, 1989). A second strategy is to derive them from commercial media sold for vertebrate cell cul- tures and modified through addition of mineral salts in oder to raise their osmolarity. As results obtained so far showed no difference between de novo made media and modified commercial media, most researchers now choose the second strategy. Commercial media most frequently used are Eagle's MEM (Cousserans, 1975; Brillouet et al., 1981; Sami et al., 1991; Cornet, 1992; Toullec et al., 1992) and Hanks 199 (Li et al., 1966; Brewster and Nicholson, 1979; Mathieu, 1987; Lenoir, 1989); RPMI 1640 has also been tested (D. Doumenc, unpubl.) and Leibovitz L-15 has recently been reported to give good results (Itami et al., 1989; Kleinschuster et al., 1992; Nadala et al., 1992). Various media sup- plements have been used: purified compounds, mainly vitamins (Machii and Wada, 1989) and non-specific nitrogens (Brillouet et al., 1981; Sami et al., 1991); homologous hemolymph (Lenoir, 1989; Machii and Wada, 1989; Kleinschuster et al., 1992); fetal calf serum (Cousserans, 1975; Brewster and Nicholson, 1979; Lenoir, 1989); horse serum (Okazaki, 1975; Kingsley et al., 1987); yeast or bacterial extracts (Klan- tan et al., 1993); extracts prepared from whole animals (Perkins and Menzel, 1964; Cecil, 1969) or from spe- cific tissues such as gonads (Dosuk, 1988) and cerebral ganglia (Lenoir, 1989).

In this study, we have investigated soluble media supplements for their ability to promote the viability of primary cultures of adult marine bivalve cells.

Because of their economic importance adult Cras- sostrea gigas oysters were chosen as material for study.

Heart tissue yielded the most easily obtained and most consistently non-contaminated cultures and, for this reason, the study of media supplements was devot- ed to this tissue. Primary cultures were initiated with the explant method, by spontaneous migration of the cell types contained in the ventricle and auricles: epi- cardiac cells, myocardiac cells, connective cells and hemocytes. In order to get rid of the explants and to cultivate only isolated cells, explants were discarded after 1 to 2 weeks of explant culture and cells that had migrated out of the explants and were adhering to the substratum were transfered to new culture vessels, covered with fresh medium and used to test various culture media.

In preliminary experiments the use of Leibovitz L- 15 was shown to significantly enhance the survival time of heart primary cell cultures as compared with RPMI 1640 (Coulon, 1993). We thus selected L-15 and mod- ified it by addition of mineral salts in order to raise its osmolarity to 1100 mosmol/l, to adjust its Na/K ratio to 37 and its Ca 2+ and Mg 2+ concentrations respective- ly to 6 mM and 25 mM (Coulon, 1993). Since in the presence of cells we measured no significant decrease in the concentrations of amino-acids and galactose of this medium (Coulon, 1993), we made the assumption that the culture's viability might not be limited by the consumption of substrates provided by the medium, but rather by the absence of growth-promoting sub- stances. According to the hypothesis that most mecha- nisms governing animal cellular viability are universal (Jacobs, 1990), we decided to test the effects of soluble growth-promoting factors of vertebrate origin.

Materials and methods

Adult Crassostrea gigas oysters from the Marennes- O16ron area of the French Atlantic coast were pur- chased year-round, opened and hearts (ventricle plus auricles) were dissected out of the pericardial cavities with sterile scissors and tweezers. In order to eliminate contamination, the excised hearts were subjected to three 1 hour rinses in a sterile "wash solution" consist- ing of artificial seawater (Sigma, 34 g/l) supplemented with broad spectra antifungal and antibacterial com- ponents: 0,2/zg/ml fungizone (Squibb), 50 units/ml nystatin (Gibco), 100 units/ml penicillin G-100/zg/ml streptomycin (Gibco), 200 units/ml polymyxin B (Pfiz- er), 10 units/ml bacitracin (Martinet) and 200/zg/ml solnicol (Synthelabo). Decontaminated hearts were then stored up to three weeks at 4~ in 10% "wash

1Ii

solution" plus 90% modified Leibovitz L-15 medium. Homologous hemolymph was prepared between Octo- ber and March: hemolymph was aspired with sterile needle and syringe from the pericardial cavity of at least six animals, pooled and centrifuged at room tem- perature at 200 g for 10 minutes. The cellular pellet was discarded, and the supernatant was inactivated by a 30 minute treatment at 56~ then filtered through a 0,22 #m filter (Analypore) and stored at -20~

Cell culture

Hearts were transferred to plastic Petri dishes, flooded with "wash solution" and minced into 1 mm 3 pieces. The pieces were rinsed with "wash solution" to remove debris, placed to adhere onto the bottom of plastic 25 cm z tissue culture vessels (Coming), and covered with 1,5 ml of medium: Leibovitz L-15 commercial formu- la (Seromed), modified by addition of NaCI (20,2 g/l), KCI (0,54 g/l), CaCI2 (0,6 g/l), MgSO4 �9 7H20 (1 g/l) and MgC12 �9 6H20 (3,9 g/l) (Merck), and 100 units/ml penicillin G and and 100 ttg/ml streptomycin (Gib- co) in order to avoid microbial development, sterilized by filtration through a 0,22 #m filter (Analypore) and stored at 4~ final pH was 7,0 and osmolarity 1100 mosmol/1. For experiments, this modified Leibovitz L- 15 medium was either used alone or supplemented with 10% (v/v) fetal calf serum (purchased from PaaGmbh, decomplemented by a 30 minute treatment at 56~ and stored at -20~ or homologous hemolymph. Explant primary cell cultures were incubated 7 to 14 days at 20~ then the suspension was removed and the pieces (explants) were detached one by one using a pipet tip; cells that had migrated out of the explants and were adhering to the bottom of the flasks were trypsinised with 1 ml of a dissociating solution, consisting of 0,05% trypsin (Seromed), 0,125% EDTA (Sigma) and 1 mg/ml glucose (Sigma) in artificial seawater (Sigma, 34 g/l) adjusted to pH 7,25. After 10 minutes incuba- tion at room temperature, trypsinisation was stopped by diluting the dissociating solution with 2 ml modi- fied Leibovitz L- 15 supplemented with 10% (v/v) FCS, cells were scrapped with a Costar cell scrapper and the suspension was centrifuged at room temperature at 200 g for 10 minutes. The ceils were resuspended in fresh culture medium, transferred to new tissue culture ves- sels and used for experiments. Viable cell density was estimated by counting of cells excluding trypan blue dye (Sigma) on a Malassez type hemacytometer. As the number of cells in these subcultures gradually decreas- es with time, the designation secondary cultures stricto

sensu is not appropriate and we still designate them as primary cultures.

MTT reduction assay

The MTT reduction assay is an enzymatic viability test, based on the reduction of a yellow tetrazolium salt (MTT) into blue formazan by active mitochondria; it was first developed by Mosmann in 1983 for screening lymphocytes stimulation and we adapted it to marine bivalve cell viability testing (Coulon, 1993). A stock solution was prepared by dissolving the MTT chemical (Sigma) in artificial seawater at 5 mg/ml final concen- tration, filtering it through a 0,22 #m filter (Analypore) and storing it at 4~ For assays, 10 #1 of this MTT stock solution was added directly to 100/zl of cell sus- pension in wells of a 96 well microplate (Nunclon); the plate was incubated for 6 hours at 20~ the formazan produced by the cells was then dissolved by adding to the wells 100 #1 of isopropanol (Merck) containing 0,04N HCI (Merck) and by shaking the plate for 10 minutes at 20~ absorbances at 580 nm were mea- sured in an ELISA-reader (Dynatech MR 300) with a reference wavelength of 630 nm (in the following des- ignated OD 580 nm/ref 630 mm); the ELISA-reader was connected to a HW-Packard compatible PC com- puter and data were transferred to Lotus 123 where their analysis was performed.

Multifactorial screening assays

Soluble factors known to influence the in vitro via- bility of mammalian, insect or fish cells were listed and grouped in five families: (1) hormones and pep- tidic mediators: bovine insulin (Pentex-Miles), hydro- cortisone, oestradiol-r putrescine (Sigma); (2) non- specific mitogens: type IV concanavalin A, PokeWeed Mitogen, E. Coli lipopolysaccharide, Multiplication- Stimulating-Activity (Sigma); (3) polypeptidic growth factors: rh bFGF (British Biotechnology), rh EGF and rb PDGF BB (Upstate Biotechnology), rh TGF/31 (Oncomembrane); (4) protective agents: aprotinin and fetuin (Pentex-Miles), catalase and gluthation-GSH (Sigma); (5) tipids and lipidic mediators: Ex-Cyte VLE and Egg Yolk Extract (Pentex-Miles), myo-inositol and prostaglandin F2c~ (Sigma).

The objective of this screening phase was to detect factors or interactions of factors having an influence on the in vitro viability of oyster cells. Complete facto- rial designs of experiments were built, in order to test all intra-family combinations of the factors (Box and

112

Table 1. Experiment notation and actual treatment for a full factorial design for four factors: A, B, C, D, tested at two levels: (-1) and (+1)

Experiment Notation Actual Treatmentl

number A B C D

1 1 (-1) (-1) (-1) (-1) 2 a (+1) (-1) (-1) (-1)

3 b (-1) (+1) (-1) (-1) 4 ab (+1) (+1) (-1) (-1)

5 c (-1) (-1) (+1) (-1) 6 ac (+1) (-1) (+1) (-1)

7 bc (-1) (+1) (+1) (-1) 8 abc (+1) (+1) (+1) (-1)

9 d (-1) (-1) (-1) (+1)

10 ad (+1) (-1) (-1) (+1) 11 bd (-l) (+1) (-1) (+1)

12 abd (+1) (+1) (-1) (+1) 13 cd (-1) (-1) (+I) (+1) 14 acd (+1) (-1) (+1) (+1) 15 bcd (-1) (+1) (+1) (+1) 16 abcd (+1) (+1) (+1) (+1)

Wilson, 1951). The numbers of factors that could be reasonably screened per experiment was restricted to four and their concentrations to two pre-defined levels: a low concentration, noted (-1), and a high concen- tration, noted (+4). All main effects and interactions within each family of 4 factors were investigated, so the total number of combinations that were tested per experiment was 24 ie 16. Notations of these combina- tions are reported in Table 1.

For each family, cells from the same inoculum cul- ture (explant cultures grown in modified Leibovitz L- 15 medium supplemented with 10% FCS) were seed- ed at about 40 000 cells/well (1,3 �9 105 cells/cm 2) on duplicate 96-well microplates (Nunclon), in 60/zl of modified Leibovitz L-15 medium supplemented with 10% (v/v) homologous hemolymph. When the cul- tures were established, after 1 day of incubation, 40 #1 of stock solutions of the factors to be tested were added to the wells (final volume: 100 #l/well): 3 repli- cate cultures (with cells) and 3 blanks (without cells) were made for each of the 16 combinations. The first plate was read after 2 days and the second after 6 days of incubation: viability of the cultures was quan- tified with the MTT reduction assay. OD 580 nm/ref 630 nm were analyzed according to the method of Yates: for each combination, an individual response

was calculated as the mean of the triplicate cultures corrected for differences in media absorption by sub- traction of the mean of the respective blanks; a general response was calculated as the mean of the 16 individ- ual responses; the effect of each factor was calculated as the difference between the 8 individual responses where the factor was at its high concentration (+1) and the 8 individual responses where the factor was at its low concentration (-1): if this difference was negative, variation of the factor between its low and its high concentration was considered inhibitory; if this differ- ence was positive, variation of the factor between its low and its high concentration was considered stim- ulatory. The effect of each interaction was calculated using the same procedure, the sign of each individu- al response resulting from the multiplication of each corresponding factor's sign. Statistical significance of the calculated effect was assayed with the Fisher test: the ratio between "due-to-factor" variance and residu- al intrinsic variance was calculated and compared with tabulated Fisher values; p > 0,20 was considered sta- tistically non-significant (NS); 0,05 < p < 0,20 slightly significant (S*); 0,01 < p < 0,05 significant (S**); p < 0,01 very significant (S* * *).

All factors and interactions of factors having a sig- nificant positive effect on the viability of the cultures were selected. Separate dose-effect experiments were then carried out, in oder to determine optimal concen- trations to be used for each selected factor.

Design of a new, concentrated, supplement. Test of its effect on the viability of the cultures as compared with homologous hemolymph and FCS

Results obtained with the screening assays were used to design a new supplement, ten times concentrated, com- pounding all the factors which variations of concentra- tions had proven to have significant positive effects on the viability of the cultures. Stored at -20~ it is in the following designated (FP1).

Using the MTT reduction assay, the effects of 10% final concentration of this (FP1), of homologous hemolymph and of FCS were tested: cells from explant cultures grown in modified Leibovitz L-15 medium supplemented with 10% FCS were seeded at about 50 000 cells/well (1,7 x 105 cells/cm 2) in a 96-well plate in 80 #1 of modified Leibovitz L-15 medium; when the cultures were established, after 1 day of incu- bation, the supplements to be tested were added to the wells (final volume: 100/zl/well); after 6 days of incu- bation, the viability of the cultures was quantified with

the MTT reduction assay; mean OD 580 nm/ref 630 nm and standard deviations were calculated and compared using the Student-Fisher test, p < 0,05 being considered statistically significant. The effects of (FP1) and FCS were then more specifically compared: the influence of varying cell seeding density was investigated; the effects of interactions between (FP1) and FCS were tested in the range 1% to 20% final concentrations, using cells adapted to low-supplementation conditions (incubated in modified Leibovitz L-15 medium sup- plemented with only 1% (FP1) and 1% FCS during the period of migration from the explants).

The development of cell cultures established in the presence or in the absence of (FP1) supplement was observed over 100 days: their morphology was checked, their viable cell densities were estimated by counting of cells excluding trypan blue dye, and their cellular contractile activities were quantified by calcu- lating the regular beating frequency of cells that had been filmed on video tape.

Results and Discussion

Factors of vertebrate origin and mutual interactions that improve the in vitro viability ofCrassostrea gigas cell cultures

The MTT reduction assay is a viability test that has already been used to optimize mammal cell culture media, namely for CHO cells (Rexen et al., 1992). There is as yet no other report of its use to optimize marine invertebrate cell cultures. Compared with aver- age intensity of signals read for mammalian cell types (usually over 0,1), OD 580nm/ref630nm read for oys- ter's heart cells are much lower, averaging 0,05; they however still remain above the threshold of detection of the Dynatech MR300 microplate reader. Advantages of this test are that it is quantitative, miniaturized and semi-automated. It provides a global measure of the number of viable cells and of their in vitro metabol- ic activity; cellular metabolic activation can thus be detected even in the absence of proliferation.

Using this test for multifactorial screening assays, we have demonstrated that several soluble factors of vertebrate origin have a significant influence on the viability of Crassostrea gigas heart cell cultures (Table 2). Separate dose-effect experiments indicated trends that confirmed the results of the multifactori- al screening assays, but except for insulin (Fig. 1) and prostaglandin F2a (Fig. 2), statistical significance

113

OD 580 nm / ref 630 nm

0,07 0.06

0.04

0.03 0.02

O.OI

0 0 0.2 2 20 50

Insulin concentration (l~g/rnl)

Fig. 1. Results of the insulin dose-effect experiment. Viability is quantified after 6 days of incubation, with the MTT reduction assay. Each data point (OD 580 n m / ref 630 nm) represents the mean 4- standard deviation of triplicate cultures, seeded at 1,7.10 s cells/cm 2 in modified Leibovitz L-15 supplemented with 10% hemolymph.

OD 580 nml ref630 nm

0.I2 0.1

0.08 0.06

0.04

0.02 0

1o4 10 -7 10 -6 10- 5 5.10- J

Pro~ae, landin b'2cc eoncenn~fion (M)

Fig. 2. Results of the prostaglandin F2c~ associated with 0,01% Ex-Cyte VLE dose-effect experiment. Viability is quantified after 6 days of incubation, with the MTT reduction assay. Each data point (OD 580 nm/ re f 630 nm) represents the mean • standard deviation of triplicate cultures, seeded at 2.105 cells/cm 2 in modified Leibovitz L-t5 supplemented with 10% hemolymph.

could not be proved and optimal concentrations to be used for each factor could not be determined. This fail- ure shows that the multifactorial assay appears to be a better tool for medium optimization than unifacto- rial dose-effect assays, partly because of its elevated number of replications (24 versus 3) and its associated mathematical analysis of results that makes it possible to pick up differences in viability-promoting activities that are quantitatively very small but still statistically significant.

Among the hormones and peptidic mediators test- ed, the use of insulin or hydrocortisone enhances after 2 days the viability of the cultures (Fisher's test indi- cates respectively p < 0,12 and p < 0,17); the interac- tion of 50 #g/ml insulin with 10 -5 M hydrocortisone has after 6 days a negative effect (p < 0,06) (Table 2). Insulin-like peptides were first discovered in 1923 in Mya arenaria and have since been shown to be present in digestive tissues and nervous ganglia of marine as well as freshwater bivalves and gastropods (Lenoir, 1989). No short term in vitro effect of 10 -s to 10 -5 M

114

Table 2. Results of the multifactorial screening assays

Family Factor Concentrations tested Effects after 2 days Effects after 6 days (-1) (+1) Separately Mutual Separately Mutual

interactions interactions

Hormones (A) bovin insulin 5/~g/ml 50 #g/ml S. + NS (A) ~ S* and (B) hydrocortisone 10 -7 M 10 -5 M S* + NS NS (B) peptidic (C) oestradiol fl 10-l0 M 10 -8 M NS, NS mediators (D) putrescin 10 -5 M 10 -3 M NS NS

(A) concanavalin A 1 #g/ml 25/~g/ml NS NS Non-specific (B) Poke Weed Mitogen 1/zg/ml 50 #g/ml NS NS NS NS mitogens (C) LPS 1/zg/ml 50/zg/ml NS NS

(D) MSA 1 ng/ml 50 ng/ml NS NS

(A) bFGF 1 ng/ml 50 ng/ml NS NS (A) ~ S, + Polypeptidic (B) EGF 0.1 ng/ml 25 ng/ml NS NS S** + growth factors (C) PDGF 0.1 ng/ml 10 ng/ml NS S** + (C)

(D) TGFfl 0.01 ng/ml 1 ng/ml NS NS

(A) aprotinin 1/~g/ml 100/~g/ml NS NS Protective (B)fetuin 0.05 mg/ml 1 mg/ml NS NS S** - NS agents (C) catalase 1/zg/ml 20/~g/ml S** + S* * * +

(D) glutathion-GSH 0 mM 0.25 mM NS NS

(A) Ex-Cyte VLE 0.01% 0.1% NS S** - (A) ~ S** + Lipidic (B) Egg Yolk Extract 0.1% 1% S* + NS NS ] mediators (C) myo-inositol 1/tg/ml 25 #g/ml NS NS /

(D) prostaglandin F2a 10 -7 M 10 -5 M NS S** - (D)

Each combination is tested in triplicate cultures. Viability is quantified with the MTT reduction assay, after 2 days and after 6 days of incubation. Effects are NS: non-significant; S*: slightly significant (0,05 < p < 0,2); S**: significant (0,01 < p < 0,05); S* * ,: very significant (p<0,01)

vertebrate insulin could be detected on the rate of DNA synthesis in Mytilus edulis mantle cells after a 6 hour treatment (Mathieu, 1987). However, we have shown in a separate dose-effect experiment (Fig. 1) that a 6 day in vitro incubation of Crassostrea gigas heart cells with 50 #g/ml bovine insulin induces a 25% rise in cellular viabil i ty (p < 0,05). In the long-term, supra-

physiologic doses of insulin could have on molluscan cells similar in vitro posit ive effects as on vertebrate cells, modulat ing the transmembrane transport of glu- cose and amino-acids (Czech, 1989). Dosuk advised in 1988 to use 10 to 100/~g/ml androgens for the in vitro culture of cells of pearl-producing Pinctada, Isog- nomon, Pteria, Pinna, Haliotis and Atrima bivalves. We have observed that, in association with 50 #g/ml insulin, 10 -4 M hydrocort isone (dissolved in 1% final non-toxic concentration ethanol) posit ively influence

the in vitro viability of Crassostrea gigas heart cells. As for vertebrate cells, steroids could act on bivalve cells by modulating the cellular receptivity to growth factos (Maurer, 1986).

We have not detected any significant influence of non-specific mitogens such as concanavalin A, PokeWeed Mitogen, E. Coli l ipopolysaccharide and

Mult ipl icat ion-Stimulat ing-Activi ty on the viabili ty of the cultures, neither after 2 nor after 6 days of incuba- tion (Table 2).

A recent discovery of an EGF-l ike factor in the tissues of Mytilus edulis mussels (Odintsova et al., 1992) has raised the question of the possible presence in bivalve tissues of vertebrate polypept idic growth factors-like substances. But up to now no experiments on the in vitro effects of growth factors of vertebrate origin on bivalve cells have been reported. We have

for the first time shown that human PDGF, human EGF and the interaction of human PDGF with human bFGF, while without effects after 2 days, have after 6 days sig- nificant positive effects on the viability of Crassostrea gigas heart cell cultures: (p < 0,05) (Table 2). As in vertebrate cells, these growth factors could act as com- petence factor (PDGF) and progression factors (EGF and bFGF) to stimulate the transition of cells from the G0/G1 to the S phase of the cell cycle (Hamet et al., 1989).

As regards the protective agents tested, we have shown that whereas fetuin has after 6 days a signif- icant negative effect (p < 0,02), catalase, an enzyme which converts H202 to H20 plus O2, has a signif- icant positive effect after 2 days (p < 0,05), that is confirmed after 6 days (p < 0,01)(Table 2). Marine bivalves are known to be able to produce oxygen free radicals, both in vivo, in microsomes of Mytilus edulis digestive gland as a response to the presence of pol- lutants (Garcia-Martinez et al., 1991) and in vitro, in Ostrea edulis and Crassostrea gigas hemocytes as a response to the presence of parasites or zymozan parti- cles (Hervio, 1992). They contain free radicals detox- ication enzymes such as superoxide-dismutase, cata- lase, gluthation-peroxidase and DT-diaphorase, which are mostly located in digestive gland microsomes (Porte et al., 1991; Narbonne, 1991). We have shown that supplementing the medium with 20 #g/ml catalase significantly improves the in vitro viability of Cras- sostrea gigas heart cells; its in vitro positive effects on the viability of oyster heart cell cultures could be explained by an improvement of the cellular protec- tion from oxidative damage. Machii and Wada advised in 1989 to add 50/zg/1 gluthation-GSH to the culture media of mantle cells of pearl-producing Bivalves. Up to now, marine invertebrate cells have always been incubated under gaseous atmospheres containing about 20% oxygen, with a resulting corresponding medium dissolved oxygen concentration of about 0,216 mM, and the composition of gaseous atmosphere has so far never been systematically investigated. High oxygen concentrations might actually favor the generation of oxygen-derived free radicals, which are known in ver- tebrate cells (Swartz, 1973) to induce peroxidations, fatty acids degradation, cellular membrane desorga- nization and DNA and protein lesions, thus finally causing cellular death.

We have shown that lipids and lipidic mediators influence the viability of Crassostrea gigas heart cell cultures: as presented in Table 2, after 2 days, Egg Yolk Extract (commercial solution, Pentex-Miles) has a sig-

115

nificant positive effect (p < 0,06); after 6 days, while separately both supplements have significant negative effects (p < 0,01), the interaction of 0,1% Ex-Cyte VLE (commercial solution, Pentex-Miles) with 10 -5 M prostaglandin F2c~ has a significant positive effect (p < 0,05). A separate dose-effect experiment (Fig. 2) showed that after 6 days, prostaglandin F2c~, when associated with only 0,01% Ex-Cyte VLE, exerts at concentrations higher than 10 -7 M a significant neg- ative effect (p < 0,05): interaction of high concen- trations of Ex-Cyte VLE and of prostaglandin F2a seems to be necessary to enhance the culture's viabil- ity. Investigations on the culture of insect cells have proven that invertebrate cells have specific needs for lipids, especially for polyunsaturated fatty acids and sterols (Goodwin, 1991). As a matter of fact, suc- cessful uses of lipid-containing supplements for the culture of marine invertebrate cells have already been reported: whole egg ultrafiltrate for Spisula solidisim- ma clam heart cells (Cecil, 1969), egg yolk extract for Mytilus edulis mantle cells (Cornet, 1992), cholesterol for pearl-producing bivalve mantle cells (Machii and Wada, 1989). Our results confirm that the viability of bivalve cell cultures is improved when the medium is supplemented with a mixture of commercial lipids and lipidic mediators: Ex-Cyte VLE, Egg Yolk Extract and prostaglandin F2a. This mixture provides cholesterol (of which 95% HDL-cholesterol), phospholipids (64% phosphatidylcholin, 19% phosphatidylethanolamin), fatty acids (of which 19% polyunsaturated, most- ly (n-6): 54% linoleic acid, 0,13% only arachidon- ic acid) and prostaglandin F2a. Biochemical analysis of marine bivalve tissues points out the high concen- trations of sterols (of which 40 to 60% cholesterol), phosphatidylethanolamine (amounting to 25 to 35% of total phospholipids) and (n-3) polyunsaturated fat- ty acids (PUFA), especially 22:6 w 3 and 20:5 w 3 fatty acids (Voogt 1983). These (n-3) PUFA, typical for marine organisms, can not be provided by verte- brate sera such as fetal calf serum which has a much higher content of (n-6) than (n-3) PUFA; to our knowl- edge, they have yet never been tested as components of marine invertebrate cell culture media. Believing that they might constitute major lipid requirements for these cells, we intend in the near future to test their effects on the viability of Crassostrea gigas heart celt cultures.

116

OD 580 nm / re f630 nm

0.1

0.09

0.08

0.07

0.06

0.05

0.04

0.03

0.02

0.01

0

none 10% 10% (FP1) 10% 10% FCS 10% 10% FCS + hemolymph hcmolymph hcrnolymph 10% (FP1)

+ 10% + 10%FCS ( ~ 1 )

medium supplement

Fig. 3. Effects of the new concentrated supplement (FP1), of homologous hemolymph and of FCS. Viability is quantified after 6 days of incubation, with the MTT reduction assay. Each data point (OD 580 nm / ref 630 nm;) represents the mean -4- standard deviation of 9 replicate cultures, seeded at 1,7.105 cells/cm 2 in modified Leibovitz L-15. Differences from non-supplemented cultures are NS: non-significant; S, : significant, p<0,01; S**: very significant, p<0,001.

viability

250 -

200 -

150 -

100-

50 �84

~dium supplement

ffPl) + IO%FCS

1)

95 520

Fig. 4. Effects of (FP1) and of FCS as a function of varying seeding density. Viability is quantified after 6 days of incubation~ with the MTT reduction assay. The viability measured in non-supplemented medium is referred to as 100. Each data point represents the mean -4- standard deviation of triplicate cultures seeded in modified Leibovitz L-15.

viab~ty

250

200

150

IOO

5o

o

(FPI) FCS

F /g , 5.

[

I 10 20 1 10 20 I 10 20 ~6 I 1 1 10 10 10 20 20 20

medium supple~rtem

Compared viability-promoting activities of (FP1) ad of FCS. Viability is quantified after 6 days of incubation, with a MTT reduction assay slightly modified in order to prevent optical bias caused by interference of MTT response with high concentrations (>10%) of FCS: before the assay, the microplate was centrifuged for 10 minutes at 200g and supematants were replaced by 110/zl of MTT-containing modified Leibovitz L-15 medium. The viability measured in medium supplemented with only 1% (FPI) and 1% FCS is referred to as 100. Each data point represents the mean -t- standard deviation of triplicate cultures, seeded at 1,7.105 cells/era 2 in modified Leibovitz L-15.

Effects of the resulting new concentrated complex sup- plement

We have designed a new complex supplement, named (FP1), which contains all the soluble factors which, alone or in mutual interaction, were shown to sig- nificantly enhance the viability of Crassostrea gigas heart cell cultures: factors were prepared at ten time their effective concentration in modified Leibovitz L- 15 medium and pooled, the resulting concentrated complex supplement being stored frozen at -20~ As described in Table 3, (FP1) compounds hormones (insulin and hydrocortisone), polypeptidic growth fac- tors (EGF and PDGF), an antioxidant (catalase) and lipids and lipidic mediators (provided by a mixture of Egg Yolk Extract, Ex-Cyte VLE and prostaglandin F2a).

As measured with the MTT reduction assay, this supplement has a positive effect on the viability of one-week old oyster heart cell cultures: supplementing the modified Leibovitz L- 15 medium with 10% homol- ogous hemolymph has no significant effect, whereas supplementing it with either 10% (FP1) of 10% FCS increases the viability by about 30% (p < 0,01); sup- plementing it with 10% (FP1) combined with 10% FCS doubles (p < 0,001) the viability (Fig, 3). The viability-promoting activities of 10% (FP1) and of the

117

combination of 10% (FP1) and 10% FCS are observed whatever the cell seeding density (Fig, 4). Study of the interactions between (FP1) and FCS has shown that, compared with low-supplemented medium containing only 1% (FP1) and 1% FCS, the use of media supple- mented either with 20% (FP1) and 1% FCS or with 1% (FP1) and 10% FCS double (p < 0,01) the viability of the cultures, and that there are no significant dif- ferences between the positive effects of these media and of media supplemented either with 10% (FP 1) and 1% FCS or with 10% (FP1) and 10% FCS (Fig. 5). All media containing 20% FCS have strong negative effects (p < 0,01) compared with the media containing 10% (FP1), 20% (FPI) or 10% FCS (Fig. 5). We thus conclude that (FP1) and FCS have at 10% final con- centrations similar positive effects on the viability of primary cultures of oyster heart cells; the use of either 20% (FP1) or of the combination of 10% (FP1) and 10% FCS increases their viability, whereas the use of 20% FCS is toxic. In oyster cell cultures, (FP1) could play the role of a substitute for FCS, having similar positive effect at 10% final concentration and being less toxic at 20% final concentration.

These results show that, whereas taken separate- ly, each constituent of the complex (FPI) supplement exerts a positive effect that is statistically significant but of low intensity, increasing the culture's viability by less than 5%, pooled into (FP1), these factors exert a positive effect of much higher intensity, increasing the culture's viability by 30%, up to 100% when com- bined with FCS. As already established for vertebrate and insect cell cultures, it demonstrates for the first time the existence in marine invertebrates cell cultures of positive synergies between hormones, polypeptidic growth factors, antioxidants and lipids provided by the medium. Multifactorial experiments may thus be more appropriate than unifactorial experiments to optimize culture media for marine invertebrate cells.

Cell cultures established in modified Leibovitz L- 15 medium supplemented with the combination of 10% (FP1) and 10% FCS were maintained viable over three months in vitro: at 102 days, they still displayed net- works of long fibroblastoid-type cells (Fig. 6), assumed to be functional cardio-myocytes as demonstrated by the preservation of their regular contractile activity: 23 • 1 beatings per minute (n = 3). The MTT reduc- tion assay measures mitochondrial activity; it thus can detect cellular activation even in the absence of proliferation and may not accurately reflect a growth promoting activity. Preliminary tests, based on direct countings of cells excluding trypan blue dye, have indi-

118

Table 3. Composition of (FPI), the new concentrated complex supplement

Hormones

insulin 500 #g/ml hydrocortisone 10 -3 M

Polypeptidic growth factors

EGF 250 ng/ml PDGF 100 ng/ml

Protective agents

catalase 500 #g/ml

Lipids and lipidic mediators

Ex-Cyte VLE 1% Egg Yolk Extract 10% providing: cholesterol 0,134 g/l

>95% HDL-cholesterol triglycerides 0,530 g/1 phospholipids 0,351 g/1

t 63,8% phosphatidylchlorin 4,6% lysophosphatidylcholin 12,8% sphingomyelin 18,8% phosphatidylethanolamine

fatty acids 0,967 g/l 19,3% polyunsaturated (mostly (n-6))

prostaglandin F2a 10-7M

(prepared in bulk and stored frozen, it is diluted ten times when used in cell cultures.)

cated in the cultures supplemented with 10% (FP1) and 10% FCS a transient period of cell proliferation: viable cell densities increased by 35 to 50% during the second month of culture, then returning at three months to the level reached at one month (Coulon, 1993). As a comparison, cell cultures established in non-supplemented medium did not contain any func- tional cardio-myocytes, but only rounded cells whose numbers decreased steadily with time. For practical reasons, we unfortunately had to stop all the cultures at 102 days, so we do not know what further devel- opment they would have had. In the near future, we will repeat these experiments of long term cultures and complete the direct countings of cells with cytofluo- rimetric analysis of their DNA content, in order to distinguish between healthy cells and degraded cellu-

lar bodies that still posess an intact membrane and so still have the ability to exclude trypan blue dye.

Conclusions

As proven by its performances in Crassostrea gigas heart cell cultures, our new concentrated supplement (FP1) might be more appropriate than classical FCS for adult marine bivalve cell cultures; results of pre- liminary tests we have done with cells derived from Crassostrea gigas gills are in support of this hypothe- sis but more tests with cells derived from other bivalve sPecies are needed for confirmation.

With the aim to further improve the viability of bivalve cell cultures, the composition of this supple- ment should be optimized, using multifactorial assays

Fig. 6. Beating, fibroblastoid-type cells in a 102 days culture from Crassostrea gigas heart, in modified Leibovitz L-15 supplemented with 10% (FP1) and 10% FCS. (scale bar: 10 #m)

to test the interactions between more specific growth factors, for instance extracts from Mytilus edulis cere- bral ganglia (Toullec et al., 1988) and EGF-like factors from Mytilus sp. (Odintsova et al. 1992). Antioxi- dants and specific lipids extracted from marine algae or invertebrates should also be screened. Concentra- tions should be optimized, in order to reduce the cost of medium. New studies should also be undertaken, focusing on physicochemical parameters (temperature, pH, osmotic pressure) and environmental parameters of the cultures (substratum, composition of gaseous atmosphere).

Acknowledgements

This work was supported in part by contract n ~ AQ-1- 264-FP from the Commission of the European Com- munities (EA.R. Research Programme in the Fisheries Sector), and was performed with the technical assis- tance of Sylvie Miossec, Bertin & Cie. We thank Pr. Echalier, Universite Paris VI, and Dr. Duval, CNRS, for the advice they provided us with along all the course of the work.

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Address for offprints: Dominique Doumenc, Laboratoire de Biolo- gie des Invert6br6s Marins CNRS URA 699, Mus6um National d'Histoire Naturelle, 57 rue Cuvier 75005 Paris, France.