Cell Cultures from the Symbiotic Soft Coral Sinularia Flexibilis

Downloaded from orbit.dtu.dk on: Oct 03, 2021 Cell cultures from the symbiotic soft coral Sinularia flexibilis Khalesi, Mohammad K.; Jiménez, Natalia Ivonne Vera; Aanen, D. K.; Beeftink, H. H.; Wijffels, R. H. Published in: In Vitro Cellular & Developmental Biology - Animal Link to article, DOI: 10.1007/s11626-008-9128-7 Publication date: 2008 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Khalesi, M. K., Jiménez, N. I. V., Aanen, D. K., Beeftink, H. H., & Wijffels, R. H. (2008). Cell cultures from the symbiotic soft coral Sinularia flexibilis. In Vitro Cellular & Developmental Biology - Animal, 44(8-9), 330-338. https://doi.org/10.1007/s11626-008-9128-7 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. In Vitro Cell.Dev.Biol.—Animal (2008) 44:330–338 DOI 10.1007/s11626-008-9128-7 Cell cultures from the symbiotic soft coral Sinularia flexibilis Mohammad K. Khalesi Received: 7 February 2008 /Accepted: 22 May 2008 /Published online: 26 July 2008 / Editor: J. Denry Sato # The Author(s) 2008 Abstract The symbiotic octocoral Sinularia flexibilis is a Introduction producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds Soft corals are a promising source of cytotoxic agents as demands innovative approaches, including coral cell cul- anticancer, anti-inflammatory, and analgesic compounds ture. We studied various cell dissociation methodologies (Faith et al. 1997; Ata et al. 2003; Duque et al. 2004). The and the feasibility of cultivation of S. flexibilis cells on soft coral Sinularia flexibilis (family Alcyoniidae) harbors different media and cell dissociation methodologies. symbiotic dinoflagellate algae (zooxanthellae) and produces Mechanical dissociation of coral tissue always yielded the bioactive compounds with cytotoxic, antimicrobial, and highest number of cells and allowed subsequent cellular anti-inflammatory activities (Dhu et al. 1998; Kamel and growth in all treatments. The best results from chemical Slattery 2005; Radhika et al. 2005; Chao et al. 2006;Yuet dissociation reagents were found with trypsin-ethylene al. 2006). The increasing interest in these resources for drug diamine tetraacetic acid. Coral cells obtained from sponta- development raises a conservation concern (Hunt and neous dissociation did not grow. Light intensity was found Vincent 2006). To avoid overexploitation of coral ecosys- to be important for coral cell culture showing an enduring tems, the production of the source animal or its cells is symbiosis between the cultured cells and their intracellular important. algae. The Grace’s insect medium and Grace’s modified Among the approaches for sustainable exploitation is the insect medium were found to be superior substrates. To coral cell culture with applications in fields such as confirm the similarity of the cultured cells and those in the ecotoxicology and biotechnology (Rodgers 2002). Tech- coral tissue, a molecular test with Internal Transcribed niques for cnidarian cell culture (including corals) have Spacer primers was performed. Thereby, the presence of not been developed yet. Problems were encountered with similar cells of both the coral cells and zooxanthella in cell dissociation, culture contamination, and medium different culture media was confirmed. (Frank et al. 1994; Odintsova et al. 1994;Rinkevich 1999). Due to the lack of validated aquatic invertebrate Keywords Coral cell culture . Sinularia flexibilis . cell lines, primary cell or tissue cultures for toxicology have Zooxanthellae . Identification test been used (Reviewed by Villena 2003). Cell cultures from a gorgonian octocoral scleroblasts were maintained for more than 4 mo (Kingsley et al. 1987). A review by Rinkevich (1999) indicates that between 1988 and 1998, only three studies on coral cell culture were published (Gates et al. 1992; Frank et al. 1994). In most cases, the cells did not survive more than 1 mo. Frank et al. M. K. Khalesi (*) (1994) observed that cryopreserved cell cultures after Sectie Proceskunde, WUR, approximately 1 yr were excluded by eukaryotic unicellular Biotechnion, Bomenweg 2, – 6703 HD Wageningen, Netherlands organisms. Again, between 1998 2004, there were no e-mail: [email protected] successful studies published on long term cnidarian cell CELL CULTURES FROM THE SYMBIOTIC SOFT CORAL SINULARIA FLEXIBILIS 331 cultures (Frank and Rinkevich 1999; Kopecky and pressing them with a loose-fit plastic piston. Sieved cells Ostrander 1999; Schmid et al. 1999; Domart-Coulon et al. were resuspended in 1.5 ml of SSW in 2-ml sterile eppendorf 2001, 2004) even though some encouraging reports were tubes. Cell suspensions were centrifuged (300×g, 10 min); the presented (reviewed by Rinkevich 2005). supernatant was discarded and the pellet resuspended in The failure to establish long-lasting and proliferating cell 1.5 ml 1% gentamycin–streptomycin solution (1:1) in SSW. cultures has been attributed to the lack of vital information This procedure was performed four times. regarding cell requirements and their physiology and biochemical patterns in vitro, improper comparisons between Chemical dissociation. Following rinsing, coral pieces vertebrate–invertebrate cell requirements (Rinkevich 1999), (1 mm3) were placed into eppendorf tubes (2 ml) containing as well as insufficient information about the classification one of the following dissociation reagents: trypsin-ethylene and identification of cell types (Puverel et al. 2005). To diamine tetraacetic acid (EDTA) 0.05% in SSW, pronase extend the functional survival of coral cells, cell culture 0.1% in SSW or collagenase 0.05% in SSW and then protocols have to be improved (Domart-Coulon et al. 2004). shaken at 200 rpm for 40 min, 60 min, and 2 h, respec- The principal aim of this study, therefore, was to investigate tively. Cell suspensions were centrifuged and resuspended the establishment of long-lasting cell culture from the as explained for mechanical dissociation. symbiotic soft coral S. flexibilis. Because both substrate composition and cell dissociation methods are critical in the Culture Media and Conditions - Media. Four enriched survival and prolongation of cells (Frank et al. 1994; media were used for cultivation: Dulbecco’s modified Eagle Odintsova et al. 1994; Rinkevich 1999), we examined various medium (DMEM), medium 199 (Frank et al. 1994), Grace’s media and cell-dissociation methodologies. Eukaryotic endo- insect medium (GIM) and Grace’s modified insect medium symbionts might develop in invertebrate cell cultures instead (GMIM; Hurton et al. 2005). To avoid crystallization, of the target coral cells. Hence, specific tests such as genetic supplements were not added (Frank et al. 1994). All media probes should be used to verify the nature of the cultured contained a 1% antibiotic cocktail (streptomycin–gentamycin cells (Villena 2003). Therefore, we have also used for the first 1:1). Cells in sterile sea water were used as a control. time a genetic approach to identify coral cells in culture. Culture containers. After initial attempts with 25 cm3 tissue culture flasks (T-flasks), cells were cultivated in 24- Materials and Methods well plates with 1.5 ml of medium and 1% antibiotic cocktail (streptomycin–gentamycin 1:1). Coral cell culture. All cultivation procedures (in four stages) were executed with sterile materials; protocols were Incubation conditions and culture maintenance. Cultures based on Gates et al. (1992), Frank et al. (1994), Kopecky were preserved in a climate cabinet (Snijders Scientific) and Ostrander (1999), and Hurton et al. (2005). with a constant temperature of 24° C and 5% (v/v)ofCO2 in air. Fluorescent light (80–100 μEm−2 s−1) was applied in Organism. Colonies of S. flexibilis were obtained from a 12 h light–12 h dark photoperiod. Cultures were checked Burgers’ Zoo, Arnhem, The Netherlands and were main- daily, and culture media were refilled almost once per week tained in laboratory tanks (25±0.5°C, 12 h light–12 h dark or when the media were below 0.7 ml. period, 34‰ salinity, and a pH of 8±0.5). Prior to dissociation, corals were rinsed at least ten times with Culture Analysis - Growth. Cultures were counted two sterile sea water (SSW; Instant Ocean Reef Crystals, times per week with an inverted microscope (Olympus Aquarium Systems, Sarrebourg, France) and sliced into CK40); pictures of all the treatments were taken using a 1-mm3 pieces with a scalpel. digital camera (Olympus C-3030) with a lens (Olympus U- PMTVC) and the software AnaliSYS for photo extracting Tissue Dissociation - Spontaneous. Coral samples (1 mm2) and scaling (Soft Imaging System GmbH). Three different were incubated in a 1% gentamycin–streptomycin (1:1) counting methods were used: after the dissociation process and solution in SSW

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