primmorphs from Stylissa massa cells
32 days after aggregation: a surface
(A) and a cross section (B) are shown
that can attach to a surface (basopinacocytes). Here, the totipotency of the archaeocytes
shows a deficiency, because only after the formation of a basal lamina are archaeocytes
able to form new collencytes. Hence, if the initial number of collencytes is too low, the
development of the sponge is terminated in an early stage. It is not known to what extent
collencytes survive the dissociation protocol executed by most researchers. It is also not
known whether the results of this embryologic study are representative for all sponges or
not. These are interesting subjects for further study by both marine biotechnologists and
sponge biologists.
IMMOBILISED SPONGE CELLS
A drawback of primmorphs is that their formation is slow (11 days) and the particles are
relatively fragile. Artificial immobilisation in gel supports could be an alternative.
Division of the immobilised cells can then be promoted by the use of growth stimulating
lectin phytohemaglutin, similar to that in dissociated cell cultures (Pomponi and
Willoughby, 1994). After division the cells can form an aggregate in the gel, so that cell-
to-cell contact is restored and further growth triggered. In addition, the gel provides a
solid matrix for the aggregates enabling them to develop further into axenic functional
sponges.
In order to show that immobilisation of sponge cells is possible and that immobilised
sponge cells can proliferate, some initial experiments were done with different sponges
(Halichondria melanadocia, Teichaxinella morchella, Agelas clathrodes, Spongosorites
sp. and Forcepia sp.). Dissociated cells were immobilised in a thermogelating gel.
Immobilisation conditions were very mild. Immobilisation was carried out using agarose
with a low gelling temperature (25 °C). For bioprocess purposes immobilisation is
usually done in gel beads. Gel beads can easily be kept in suspension in bioreactors up to
a holdup of 25%. In order to show effects of immobilisation on cell viability and
proliferation capacities, gel layers were made that could easily be observed
microscopically. The gel with cells was brought into multiwell-plates to form a layer of
1–3 mm thickness. These layers were covered with growth medium.
Microscopic observations revealed that many cells survived the immobilisation step.
In most experiments, both single cells and small aggregates were present and these could
be kept in an apparently viable state for at least two weeks. In addition, the gel material
often remained free of overgrowth by contaminating micro-organisms. As an example
results are given of Halichondria melanodocia. Individual cells were immobilised. After
9 days of cultivation all cells developed in aggregates due to growth, as is shown in
Figure 17.7. Cell proliferation was very similar to the development of micro-colonies that
are normally observed when immobilised micro-organisms are grown. In other
experiments aggregates of Agelas clathrodes also showed some growth during the first
week. In addition their outer appearance changed from rather irregularly shaped clumps
to spherical aggregates with a smooth surface. The best results were obtained with
Multiphase bioreactor design 520