Techniques are available to separate different cell fractions. Usually, a crude cell
suspension is put into a density gradient consisting of two or more layers with increasing
density, followed by centrifugation at ~600 g. Density gradients can be made of Ficoll or
Percoll solutions, commercially available fluids with a high density, that can be diluted
with CMF-ASW. The bands of cells that are formed at the interfaces between layers must
be carefully collected, for instance by using a pasteur pipet, and transferred to clean
CMF-ASW or sterilised seawater. In this way fractions that are enriched in specific cell
types such as archaeocytes can be obtained.
So far it has not been possible to produce a continuous cell line. Cell division was
successfully stimulated with different growth factors, but a continuous cell line could still
not be established. Only initial cell proliferation could be stimulated (Ilan et al., 1996,
Pomponi et al., 1997).
Once more, we emphasize that despite their totipotency and high proliferative capacity
in functional sponges, even archaeocytes have not been shown capable of growing in
suspension. Potential causes and possible solutions are:
- Immortal Sponge Cells are not Available
Animal cell lines from insects and mammals usually consist of cells that are immortal;
they have an unlimited capacity to proliferate. Immortal cells of mammals can be
obtained from tumour tissue. Immortality can also be induced artificially, either by
hybridisation of normal cells with immortal cells (e.g. hybridoma’s) or by subjecting the
cells to mutagenic agents such as carcinogenic compounds, viruses or radioactivity.
Sometimes immortal cells evolve spontaneously by mutation of normal cells growing in
rich media. It is unknown to us to what extent these methods have been applied to sponge
cells; no reports on successful immortalisation of sponge cells have yet been published. It
should be borne in mind here that out of a thousand attempts to establish mammalian and
insect cell lines, only a few are successful. It is therefore well worth continuing attempts
to develop immortal, continuous sponge cell lines, by using both existing techniques and
by applying the methods mentioned above to produce immortal sponge cells.
An advantage of sponge archaeocytes over other animal cells is their free-living
nature. They are not part of multicellular tissues, which may reduce the time the cells
need to adapt to life in suspension. Another advantage is that sponge cells have a high
telomerase activity. This enzyme stimulates the restoration of the telomeric ends of the
chromosomes after mitosis, which is assumed to be a crucial process for cells continuing
their division cycle. In theory, somatic sponge cells can thus maintain the mitotic cycle
longer than somatic cells of other animals. In other words they are closer to immortal
cells than most other somatic cells. However, the contrasting inability of sponge cells to
proliferate in rich media containing foetal calf serum remains a serious obstacle, because
this considerably reduces the possibility that an immortal cell line evolves spontaneously
by mutation.
- Cell-to-cell Contact Stimulates Telomerase Activity
Why do dissociated sponge cells hardly proliferate in media containing foetal calf serum?
Apparently, the media used until now to culture sponge cells do not sufficiently resemble
Multiphase bioreactor design 514