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    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Record number 333491
Title Cultivation of Marine Sponges: From Sea to Cell
Author(s) Sipkema, D.
Source Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Rene Wijffels; Ronald Osinga. - Wageningen : Detmer Sipkema - ISBN 9789085040743 - 184
Department(s) Bioprocess Engineering
VLAG
Publication type Dissertation, internally prepared
Publication year 2004
Keyword(s) secundaire metabolieten - farmaceutische producten - sponsen - kweektechnieken - secondary metabolites - pharmaceutical products - sponges - culture techniques
Categories Plant Biotechnology / Food and Bioprocess Engineering (General)
Abstract Marine sponges are one of the richest natural sources of secondary metabolites with a potential pharmaceutical application. A plethora of chemical compounds, with widely varying carbon skeletons, possessing among other anticancer, antiviral, antibiotic, antiinflammatory and antimalaria activity has been discovered. While for most metabolites their molecular mode of action is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases has been reported. Knowledge on the mode of action is one of the key factors required to transform bioactive compounds into medicines. The rich diversity in bioactive compounds from sponges has provided molecules that interfere with the pathogenesis of a disease at many different points, which increases the chance of developing selective drugs against specific targets (Chapter 2).Unfortunately, these secondary metabolites are usually present in trace amounts, and natural stocks are too small to sustain the development of widely available medicines. The development of ways to obtain large quantities of the secondary metabolites is therefore currently the most important quest. A number of biotechnological methods could potentially provide the required amount of bioactive substances. Three methods were studied in this thesis:

Ex situculture

The term ex situ culture refers to cultivation of functional sponges outside of the sea. One of the crucial issues for the ex situ cultivation of sponges is the design of a suitable growth medium. Generally sponges are regarded as particle feeders (bacteria and algae), but they are also capable of the uptake of (partly) dissolved organic carbon sources. The use of powdered substrates can be beneficial for the ex situ culture of sponges under controlled conditions, because an optimal mix of nutrients can be developed and a constant quality can be guaranteed. The ex situ growth rates of sponges cultured on these substrates could be improved, when compared to the sea, but they remain low and resulted in long-term experiments. In order to optimise the growth rate of sponges, it is important to have insight in the way that sponges grow. The suitability of three different models (linear, exponential and radial accretive growth) to describe the growth of both globose and encrusting sponges was assessed. For both morphological appearances, radial accretive growth was the preferred model to simulate the growth. The model can be a valuable tool to make a sound comparison between growth rates of different sponges. In addition, it can be used to study the quantitative effect of factors, such as pressure, light, current, age, temperature or the nutrient source or -concentration on the growth rate of sponges (Chapter 3).

Primmorphs

Primmorphs are spherical-shaped sponge-cell aggregates with a diameter of approximately 1 mm. They are formed from a dissociated cell suspension under gentle agitation and resemble buds and gemmules, which are the naturally produced asexual regeneration bodies. Primmorph formation seems to be a universal characteristic of marine sponges, as they were obtained from seven different species. By scanning electron microscopy (SEM) it was observed that the primmorphs are very densely packed sphere-shaped aggregates with a continuous pinacoderm (skin cell layer) covered by a smooth, cuticle-like structure. The latter characteristic is probably the reason why primmorphs are more robust than functional sponges and can be easily maintained for a long time. Incubation of primmorphs in a rich medium to attempt cultivation of the aggregates frequently resulted in the growth of bacterial, fungal and eukaryotic unicellular contaminants, which prevented a growth study of primmorphs. The addition of gentamycin or a mixture of penicillin and streptomycin could usually avoid bacterial contaminants, but eukaryotic contaminants were persistent. The addition of the fungicide amphotericin B or a cocktail of antibiotics (kanamycin, gentamycin, tylosin and tetracyclin) prevented the formation of primmorphs (Chapter 4).

If primmorphs are actually a kind of experimentally induced regeneration bodies, they could develop into functional sponges. When primmorphs were maintained in seawater enriched with silicate (70 or 150 µM) it was observed that they indeed produced spicules (silica-based skeletal elements) and attached to the bottom of the culture dish, which never occurred at lower silicate concentrations (4 or 25 µM). These results may be explained by available knowledge on the molecular level. Silicate is known to induce the expression of silicatein, the enzyme involved in the production of spicules, at concentrations higher than 60 µM. In addition, silicate has been found to stimulate the biosynthesis of myotrophin, which enhances the production of collagen. Collagen is well known to play an important role in both the attachment of gemmules to a substratum and their subsequent morphogenesis (Chapter 5).

Sponge-cell culture

Sponge-cell culture may be the tool to overcome the low growth rate, and the corresponding low production rate of the bioactive metabolites of functional sponges. However, the presence of large numbers of associated bacteria, fungi and unicellular organisms inside sponges has been a major obstacle in the development of sponge-cell lines. They have prevented the formation of axenic sponge-cell suspensions, and proliferating sponge cells in cell cultures were therefore looked at with suspicion.

For that reason two of prerequisites for the cultivation of sponge cells were developed:

A method to distinguish sponge cells in culture from contaminants.A method to assess the viability of cells in culture.The 18S rRNA gene is a suitable marker to identify the origin of eukaryotic cells and a genetic detection method based on this gene was developed for the sponge Dysidea avara . The 18S rRNA gene from a Dysidea avara specimen was sequenced and compared to eukaryotic 18S rDNA sequence(s) that were picked up from a proliferating cell culture that originated from a dissociated Dysidea avara specimen. This method proved to be successful to unambiguously detect whether the cells in culture were actually sponge cells or contaminants (Chapter 6).

Cell viability is an essential tool to study the effect of medium components on cell physiology. Especially in case of primary sponge-cell lines it is important to know whether slow growth is caused by a low specific growth rate or by a low viability of the cells. Trypan blue exclusion is a commonly used method to estimate the viability of cell cultures, but for unknown reasons this does not work properly with sponge cells. Therefore, a flow-cytometric viability assay, based on the combined use of fluorescein diacetate (FDA) and propidium iodide (PI) was developed. The effects of temperature, ammonium and the fungicide amphotericin B on the viability of a primary cell culture were studied as examples to assess the suitability of the test. Cell fluorescence measurements based on incubation of cells with FDA or PI, resulted in a good and reproducible estimate of the viability of primary sponge-cell cultures. It was found that the cells rapidly die at a temperature of 22 °C or higher, but that they are insensitive to ammonium concentrations up to 25 mM. Amphotericin B was found to be toxic to the cells (Chapter 7) and this could explain why no primmorphs were formed in the presence of this antibiotic.

The current technical status of different methods to produce sponge metabolites was used to study the feasibility of pharmaceuticals from sponges at a large-scale. The production of the metabolites halichondrin B and avarol by chemical synthesis, wild harvest, mariculture, ex situ culture, primmorphs, sponge-cell culture, genetic modification and semi-synthesis were compared on a technical and economical basis, as far as possible. Halichondrin B from a Lissodendoryx sp. and avarol from Dysidea avara were used as model compounds as their products are opposites with respect to their natural concentration inside the sponge. It is concluded that for avarol, which is present in a relatively high concentration, mariculture and ex situ culture could offer feasible methods to compete with currently used medicines against psoriasis. For halichondrin B, the low concentration is a bottleneck for sponge biomass-based production of the compound. A combined approach of (genetically modified) bacterial fermentation (to produce a precursor molecule) followed by a limited number of chemical steps to produce molecules that are derived from sponge chemicals will probably be the most successful method to develop medicines from sponge metabolites that are present in low concentrations (Chapter 8).
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