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

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ln th e rirsl ex perimcIlls, wc have in oc ulalCd cc lls
obtain cd fro m 1.7 dissociated venll'icles l'cr f1a sk.
But best growth results were further obtaincd when
cell s from 3.5 venll'icles were inocll'ated per f1 ask.
Indeed. as much cell s died quickly, on the first two
days, il \Vas necessary to inoculated a grc3t numbcr
of eells 10 rccover enough anchored cells. Moreover.
a grea ter number o f ancho red ec ll s provides an exlrace
llular microenvironment w llich could enhance Ihe
cc II growth and me ta boli sm 17, 13J. T hese resu lts
also suggest Ihat sOllle celllypes may relcase fa ctors
necessary for the development o f other cell types.
Such a coopera tive growth may need a greatcr
nllmbc r o f ccll s per f1ask .
Moreover, previous poly-D-Iysin coating of f1 asks
facilitated the ccii at tac hment. The examination of
cultures under light inverled micro scope did not
reveal noticeable differences between poly-D-Iys in
coated and control f1 asks on the first days. But 6 ta
7 days later in control f1a sks, cel ls started to detach
while cell s still anchored exhibited poor preserved
aspect. In pol y- D-Iysin coated f1asks, cells remained
healthy, adhesivc and growing for at least 15 days
(Figure 2). It is weil known that the extracellular
cnvironment is of great importance si nce il influences
the metaboli sm or cells, but also the cytosqueletton
structure and thus, the morphology of cells in vivo
and in vitro [1 J. Moreover, the previous coating of
f1a sks with poly-D-Iysin by enhancing the anchorage
of cell membrane to the plastic f1a sk bottom, also
promotes the ccii division [13J.
Last of ail, we supposed the presence of growth
factors or nutriti ve eleme nts in the hemolymph of
oyster [1 2] and we have modified this procedure for
primary culture of oyster heart cells by adding oyster
hemolymph to the c ulture medium. We compared
medium L- 15: sea water ( 1: 1) supplemented wi th differen
t combinations of 0, 1,5 or 10% COH and 0, 5
or 10% FCS. We found that the presence of 5 ta 10%
FCS was necessary. 1 % COH was not different From
control 0% COH. While, in 5% COH supplemented
medium. cc II growth scc mcd ta be better becausc cell
layers looked more dense, however, ce ll preserv ation
did not extend in time. Whereas. the presence of 10%
COH seemed to be taxie ror cell s, as they were more
damaged than in co mml without CGH.
lt is important to mention that propenies or CGH
are greatly affected by freezing to - 20 oc. Indeed,
improveme nt of cc II growth wit h COH di sappeared
artel' thawing but more , il became a Few damag ing
to cells. Lyophylisation and storage at 4 oC seemed
beller si nce no difference was ûbserved between
Iyophylised and fresh COH. However, the preservati
on o f suc h Iyophilised COH is unknown for very
long periods.
By transmi ss ion eleclron mi croscopy examinatioll,
different cell types could be identified in the cultures.
They werc mostly card iomyocytes (Figure 3), fibreblast-like
cell s (Figure 4) and pigmented ce ll s (Figure
5). Other cell types we re supposed to be haemocytes,
particularly granulaI' type hae mocytes were fre ­
quently observed (Figure 6).
These observ at ions revcalecl th e weil prese rved
ultrastructurc of mûst of the cullured cells. This seem
to indicate that they kcep a correct bi ological activity
since thei!" aspect \Vas similar la th ose of the cells
observed in Silu , in non di ssoc iated ventricles fixed
with g lutaraldehyde just after dissection . However,
pigmented ce Ils present in cultures were less pigmented
th an the same type of cell s observed in situ ,
indicati ng that they may have released a part of their
pigments (Figure 5). Transmi ss ion electron mi cro ­
graphs also indicate tha t some cell s degenerate with
time in cu lture . Indecd. il \Vas observed myelin
figures corresponding to the accumulation of degra ­
dation products.
The different cc ii types present in the cultures
offer many possibilities of applications of thi s
method. Particularl y, the presence of fibroblast-like
cells in these primary cell cultures allows us to plan
10 inocu late herpes- like viru s of C. gigas larvae in
these cultures. Indeed, thi s viru s hold s a major
Figure 1. Focus of growi ng fibroblasti e type ee ll s appea r on the thinl day of cu lture in 10% FCS supple mented
L·15:sea \Vater (3:2 and 2:3) medium.
Figure 2. Monolaycr of ten da ys cultured cell s in poly-D-Iysin coated Oasks and in the presence of the optim ized
med ium.
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