Contribution à l'étude de virus de mollusques marins apparentés ...

PUBLICATION 7
Journal Q/Tissue CI/lIUre Methods 16: 67-72. 1994.
© 1994 Kl/IIH'r Academie Publishers. Primed i" the Netlurlands.
Primary culture of Pacific oyster, Crassostrea gigas, heart cells
Rose-Marie Le Deuff, Cécile Lipart & Tristan Renault
lFREMER, Laboratoire de Biologie et d'Ecologie des Invertébrés Marins, Unité de Recherche en Pathologie et
Immunologie Générales. La Tremblade, France
Summary. Pacific oyster, Crassostrea gigas, is the
most economicaIly important specie to the world
sheIlfish breeding. It is important to note that infec·
tious diseases, particularly viruses, may be hazardous
for the C. gigas live-stocks. The study of these viral
di seases and the development of diagnosis method
need the establishment of in vitro methods for viral
multiplication. As no oyster œIl line is available
actually, we have developed a procedure for primary
culture of heart ceIls which could enable to study
molluscan viruses in vitro, and could also provide a
diagnosis method based on the search of eventual
cytopathogen viral effects. Cells from C. gigas ven-
Key words: Cell culture, Crassostrea gigas, Heart, MoIluscs, Oyster
1. Introduction
Among the shellfish breeding, Pacific oyster,
Crassostrea gigas, is the most economicaIly important
species (4), However, it is subject to the hazards
of infectious diseases. Noticeably, viruses were found
in association with mortalities of oysters [3, 5, 6, 8,
10, Il). To date, the study of these viruses was
limited to the electron microscopy description, and
the experimental reproduction of these diseases was
only reported in the case of a herpes-like virus (9).
Viral multiplication on cell culture was described for
none of these viruses. Indeed, as no molluscan œil
line is available, the search for viral cytopathogen
effects on homologous cell line is impossible
actually. Attempts were perfonmed in the laboratory
to inoculate a C. gigas herpes-Iike virus on fish and
insect œlllines, but we failed to obtain a viral replication
in these cells (results not shown). These observations
point out the need for homologous primary
ceIl cultures and cell Iines prepared from tissues of
the species naturaIly infected by the virus. Although,
a previous report describes the culture of C. gigas
heart cells (2), the cellS Iysed after a short period
(8- 15 days), and were not weil conserved during this
period. Thus, in this study, we describe the improvement
of a method for the dissociation of tissues and
the optimization of the culture medium. We also
report a detailed protocol for primary culture of confluent
monolayers of C. gigas heart cells. In addition,
morphologic description of Ihese cultured ceIls was
125
tricle of heart were dissociated by trypsin-EDTA
treatment and the mechanical action of a Dounce
type homogeneizer. The ceIls were inoculated in previously
poly-D-Iysin coated flasks. The optimised
culture medium was L-15 (Leibovitz) prepared three
fold concentrated. then diluted half with sea water,
this mixture was supplemented with 10% FCS and
5% C. gigas hemolymph. Different cell types could
be identified by transmission electron microscopy
analysis, as mostly cardiomyocytes, fibroblast-like
ceIls and pigmented cells, but also haemocytes were
present in the cultures.
performed and compared to the œil types observed
in vivo.
2. Materials
A. Culture media, solutions and chemicals
Poly-D-Lysin solution:
10 mg sterile poly-D-Lysin, No. 644 587'
100 ml sterile distilled water.
Store aliquots al -20 oC,
Chloride solution
105 ml, 50 oC chloride water'
15 g NaHCO" No. S8875'
365 g. caster sugar'
DistiIled water to 1.42 liter.
Store at 4 oC.
Autoc1aved sea water
Sea water used to wash oysters was filtered
through 0.45 !lm, autoc1aved and stored at
room temperature.
Sea water-tween
0.5 ml Tween 20, No. PI379'
500 ml autoc1aved sea water.
Sea water used in culture media was filtered
through 1 !lm, sterilized by filtration to 0.22
!lm and stored at 4 oC,
Trypsin-EDTA solution:
2 gll trypsin 1:250, No. 0152-13-1'
0.4 gll, EDTA, No. 20 296.291 6
8 gll. NaCI, No. S5886'