at present be considered a future option for the biotechnological production of natural
products from sponges.
A potential risk of using sponge cell or tissue cultures for metabolite production is that
the sponge cells may loose the ability to produce the target compound when grown in
axenic culture. It has been shown that in the culture of T. morchella, the cells were still
able to produce the target metabolite, even after artificially induced cell division
(Pomponi et al., 1997).
DISSOCIATION OF SPONGE TISSUE
Artificial dissociation of the sponge tissue is the first step to obtain primary cell cultures.
Both mechanical and chemical dissociation methods exist. A combination of these
usually gives the best result. The mechanical part consists of cutting the sponge tissue
into small pieces, which are then squeezed through a cloth or another fine mesh to
dissociate the tissue and separate the cells from non-cellular components such as spicules.
The resulting suspension of cells and small aggregates is collected in CMF-ASW,
artificial seawater that does not contain calcium and magnesium ions. Ca2+ and Mg2+ play
a crucial role in the binding between sponge cells, and the absence of these ions not only
causes the cell-to-cell bond to break, but also prevents the cells from re-aggregating (Ilan
et al., 1996; Pomponi et al., 1997).
After the dissociation step, the cells are treated with antibiotics and antimycotics. The
ultimate goal of this step is to obtain axenic (contamination-free) cell cultures, although
complete removal of all contaminants has proven to be a difficult task. However, if
properly used, the antibiotics will at least prevent overgrowth of the primary sponge cell
culture by microorganisms. The treatment with antibiotics is a very critical step, because
excessively high antibiotic concentrations may kill the sponge cells, while very low
concentrations will not sufficiently inhibit all contaminants. A combination of rifampicin
(a broad spectrum antibiotic) and amphotericin (an antifungal compound) has been used
successfully to obtain axenic cell cultures of Hymeniacidon heliophila and Teichaxinella
morchella. The concentrations used were 1.16 μM and 2.5 mg dm−^3 respectively.
However, dose-response relations have to be determined for every new combination of
sponges and antibiotics. A basal sponge culture medium has been described in the
literature in which primary sponge cells can be maintained for a limited period (Pomponi
et al., 1997; Pomponi and Willoughby, 1994).
SPONGE-CELL GROWTH
Of the several cell types found in sponges, archaeocytes and choanocytes most often
show mitotic activity. Hence, growth in sponges is thought to take place by proliferation
of these two cell types partially followed by functional differentiation of the newly
formed archaeocytes. Archaeocytes show better survivability in primary cell cultures than
choanocytes. The totipotent nature of the archaeocytes and their better survival in
primary cell cultures make these cells the best candidates for developing continuous cell
lines.
Marine sponges as biocatalysts 513