Skip to main content
SpringerLink
Log in

Primmorphs Cryopreservation: A New Method for Long-Time Storage of Sponge Cells

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

The possibility to cryopreserve cells allows for wide opportunities of flexible handling of cell cultures from different sponge species. Primmorphs model, a multicellular 3D aggregate formed by dissociated sponge cells, is considered one of the best approaches to establish sponge cell culture but, in spite of the available protocols for freezing sponge cells, there is no information regarding the ability of the latter to form primmorphs after thawing. In the present work, we demonstrate that, after a freezing and thawing cycle using dissociated Petrosia ficiformis cells as a model, cells viability was high but it was not possible to obtain primmorphs. The same protocol for cryopreservation was then used to directly freeze primmorphs. In this second case, after thawing, viability and the cellular proliferative level were similar to unfrozen standard primmorphs. Spiculogenesis in thawed primmorphs was evaluated by quantifying the silicatein gene expression level and by assaying the silica amount in the newly formed spicules, then compared with the correspondent values obtained in standard unfrozen primmorphs. Results indicate that the freezing cycle does not affect the spiculogenesis rate. Finally, the expression level of heat shock protein 70, a well-known stress marker, was assayed and the results showed no differences between frozen and unfrozen samples. These findings are likely to promote relevant improvements in sponge cell culture technique, allowing for a worldwide exchange of living biological material, paving the way for cell banking of Porifera.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Bischof JC, Padanilam WH, Ezzell RM, Lee RC, Tompkins RG, Yarmush ML, Toner R (1995) Dynamics of cell membrane permeability changes at supraphysiological temperatures. Biophys J 68:2608–2614

    Article  PubMed  CAS  Google Scholar 

  • Bradford MA (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Carvahlo de Souza A, Ganchev DN, Snel MM, van der Eerden JP, Vlieqenthart JF, Kamerling JP (2009) Adhesion forces in the self-recognition of oligosaccaride epitopes of the proteoglycan aggregation factor of the Marine sponge Microciona prolifera. Glycoconj J 26:457–465

    Article  Google Scholar 

  • Cattaneo-Vietti R, Bavestrello G, Cerrano C, Sarà A, Benatti U, Giovine M, Gaino E (1996) Optical fibres in an Antarctic sponge. Nature 383:397–398

    Article  CAS  Google Scholar 

  • Chernogor LI, Denikina NN, Belikov SI, Ereskovsky AV (2011) Long-term cultivation of primmorphs from freshwater Baikal sponges Lubomirskia baikalensis. Mar Biotechnol 13(4):782–792

    Article  PubMed  CAS  Google Scholar 

  • Custodio MR, Prokic I, Steffen R, Koziol C, Borojevic R, Brummer F, Nickel M, Müller WEG (1998) Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death. Mech Ageing Dev 105:45–59

    Article  PubMed  CAS  Google Scholar 

  • de Caralt S, Uriz MJ, Wijffels RH (2007) Cell culture from sponges: pluripotency and immortality. Trends Biotchnol 25:467–471

    Article  Google Scholar 

  • Duckworth A (2009) Farming sponges to supply bioactive metabolites and bath sponges: a review. Mar Biotechnol 11:669–679

    Article  PubMed  CAS  Google Scholar 

  • Freshney RI (1994) Culture of animal cells: a manual of basic techniques, 5th edn. New York, Wiley

    Google Scholar 

  • Gomot L (1971) The organotypic culture of invertebrates other than insects. In: Vago C (ed) Invertebrate tissue culture. Academic, New York, pp 41–136

    Google Scholar 

  • Grasela JJ, Pomponi SA, Rinkevich B, Grima J (2012) Efforts to develop a cultured sponge cell line: revisiting an intractable problem. In Vitro Cell Dev Biol Anim 48(1):12–20

    Article  PubMed  Google Scholar 

  • Green AE, Athreya B, Lehr HB, Coriell LL (1967) Viability of cell cultures following extended preservation in liquid nitrogen. Proc Soc Exp Biol Med 124:1302–1307

    Google Scholar 

  • Hink WF (1979) Cell lines from invertebrates. In: Jakoby WB, Pastan IN (eds) Methods in enzimology; cell culture. Academic, New York, pp 450–466

    Chapter  Google Scholar 

  • Jarchow J, Burger MM (1998) Species-specific association of the cell-aggregation molecule mediates recognition in marine sponges. Cell Adhes Commun 6:405–414

    Article  PubMed  CAS  Google Scholar 

  • Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of humantelomerase activity with immortal cells and cancer. Science 266:2011–2015

    Article  PubMed  CAS  Google Scholar 

  • Koziol C, Borojevic R, Steffen R, Müller WEG (1998) Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells. Mech Ageing Dev 100(2):107–120

    Article  PubMed  CAS  Google Scholar 

  • Kuhns WJ, Weinbaum G, Turner R, Burger MM (1974) Sponge aggregation a model for studies on cell–cell interactions. Ann N Y Acad Sci 234:58–74

    Article  PubMed  CAS  Google Scholar 

  • Le Pennec G, Perovic S, Ammar MS, Grebenjuk VA, Steffen R, Brummer F, Müller WEG (2003) Cultivation of primmorphs from the marine sponges Suberites domuncula: morphogenetic potential of silicon and iron. J Biotechnol 100(2):93–108

    Article  PubMed  Google Scholar 

  • Lichtenfels R, Biddison WE, Schulz H, Vogt AB, Martin R (1994) CARE-LASS (calcein-release-assay), an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity. J Immunol Meth 172(2):227–239

    Article  CAS  Google Scholar 

  • McMahon P (2000) System for the cell culture and cryopreservation of marine vertebrates US patent n 6,054,317

  • Miki W, Kon-ya K, Mizobuchi S (1996) Biofouling and marine biotechnology: new antifoulants from marine invertebrates. J Mar Biotechnol 4:117–120

    CAS  Google Scholar 

  • Mitsuhashi J (1989) Nutritional requirements of insect cells in vitro. In: Mitsuhashi J (ed) Invertebrates cell system applications. Vol. I. CRC Press, Florida, pp 3–20

    Google Scholar 

  • Mossman T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol 65(1–2):55–63

    Google Scholar 

  • Müller WEG, Müller IM (2003) Analysis of the sponge [Porifera] gene repertoire: implications for the evolution of the metazoan body plan. Prog Mol Subcell Biol 37:1–33

    Article  PubMed  Google Scholar 

  • Müller WEG, Zahn RK, Gasic MJ, Dogovic N, Maidhof A, Becker C, Diehl-Seifert B, Eich E (1985) Avarol, a cytostatically active compound from the marine sponge Dysidea avara. Comp Biochem Physiol 80:46–52

    Google Scholar 

  • Müller WEG, Wiens M, Batel R, Steffen R, Borojevic R, Custodio MR (1999) Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula. Mar Ecol Prog Ser 178:205–219

    Article  Google Scholar 

  • Müller WEG, Bohm M, Batel R, De Rosa S, Tommonaro G, Müller IM, Schöder HC (2000) Application of cell culture for the production of bioactive compounds from sponges: synthesis of avarol by primmorphs from Dysidea avara. J Nat Prod 63(8):1077–1081

    Article  PubMed  Google Scholar 

  • Müller WEG, Belikov SI, Tremel W, Perry C, Gieskes WC, Boreiko A, Schröder HC (2006) Siliceous spicules in marine demosponges (example Suberites domuncula). Micron 37:107–120

    Article  PubMed  Google Scholar 

  • Munro MHG, Blunt JW, Lake RJ, Litaudon M, Battershill CN, Page MJ (1994) From seabed to sickbed: what are the prospects? In: Van Soest RWM, Van Kempen TMG, Braekman JC (eds) Sponges in time and space. Balkema, Rotterdam, pp 473–484

    Google Scholar 

  • Natalio F, Mugnaioli E, Wiens M, Wang X, Schröder HC, Tahir MN, Tremel W, Kolb U, Müller WEG (2010) Silicatein-mediated incorporation of titanium into spicules from the demosponge Suberites domuncola. Cell Tissue Res 339:429–436

    Article  PubMed  Google Scholar 

  • Osinga R, Tramper J, Wijffels RH (1988) Cultivation of marine sponges for metabolic production: application for biotechnology? Trends Biotechnol 16:130–135

    Article  Google Scholar 

  • Osinga R, Tramper J, Wijffels RH (1999) Cultivation of marine sponges. Mar Biotechnol 1(6):509–532

    Article  PubMed  CAS  Google Scholar 

  • Peláez J, Bongalhardo DC, Long JA (2011) Characterizing the glycocalyx of poultry spermatozoa: III Semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates. Poult Sci 90:435–443

    Article  PubMed  Google Scholar 

  • Perry CC (1989) Chemical studies of biogenic silica. In: Mann S, Webb J, Williams RJP (eds) Biomineralization, chemical and biological perspectives. Wiley, Weinheim, pp 223–256

    Google Scholar 

  • Pomponi SA, Willoughby R, Edward Kaighn M, Wright AE (1997) Development of techniques for in vitro production of bioactive natural products from marine sponges. In: Maramorosch K, Mitsuhashi J (eds) Novel directions and biotechnology applications. Science, New York, pp 231–237

    Google Scholar 

  • Pozzolini M, Sturla L, Cerrano C, Bavestrello G, Camardella L, Parodi AM, Raheli F, Benatti U, Mueller WE, Giovine M (2004) Molecular cloning of silicatein gene from marine sponge Petrosia ficiformis (Porifera, Demospongiae) and development of primmorphs as a model for biosilicification studies. Mar Biotechnol 6(6):594–603

    Article  PubMed  CAS  Google Scholar 

  • Pozzolini M, Valisano L, Cerrano C, Menta M, Schiaparelli S, Bavestrello G, Benatti U, Giovine M (2010) Influence of rocky substrata on three-dimensional sponge cells model development. In Vitro Cell Dev Biol Anim 46:140–147

    Article  PubMed  Google Scholar 

  • Rannou M (1971) Cell culture of invertebrates other than molluscs and arthropods. In: Vago C (ed). Invertebrate tissue culture. New York: Academic Press vol 1, pp 385–410

  • Rinkevich B (1999) Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements. J Biotechnol 70:133–153

    Article  CAS  Google Scholar 

  • Rinkevich B, Blisko R, Ilan M (1998) Further steps in the initiation of cell cultures from embryo and adult sponge colonies. In Vitro Cell Dev Biol 34:753–756

    Article  CAS  Google Scholar 

  • Rottmann M, Schröder HC, Gramzow M, Renneisen K, Kurelec B, Dorn A, Friese U, Müller WEG (1987) Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II. EMBO J 6:3939–3944

    PubMed  CAS  Google Scholar 

  • Rozas EE, Albano RM, Lôbo-Hajdu G, Müller WEG, Schröder HC, Custódio MR (2011) Isolation and cultivation of fungal strains from in vitro cell cultures of two marine sponges (Porifera: Halichondrida and Haplosclerida). Braz J Microbiol 42:1560–1568

    Article  Google Scholar 

  • Sarà M, Bavestrello G, Cattaneo-Vietti R, Cerrano C (1998) Endosymbiosis in sponges: relevance for epigenesis and evolution. Symbiosis 25:57–70

    Google Scholar 

  • Saxena AK, Ramachandrani S, Dwivedi A, Sharma R, Bajpai VK, Bhardwaj KR, Balapure AK (1995) Simplified cryopreservation of mammalian cell lines. In vitro Cell Dev Biol 31 A:326–329

    Article  Google Scholar 

  • Schippers KJ, Sipkema D, Osinga R, Smidt H, Pomponi SA, Martens DE, Wijffels RH (2012) Cultivation of sponges, sponge cells and symbionts: achievements and future prospects. Adv Mar Biol 62:273–337

    Article  PubMed  Google Scholar 

  • Seibert G, Raether W, Dogovic N, Gasic MJ, Zahn RK, Müller WEG (1985) Antibacterial andntifungal activity of avarone and avarol. Zbl Bakt Hyg A 260:379–386

    CAS  Google Scholar 

  • Selvin J (2009) Exploring the antagonistic producer Streptomyces MSI051: implications of polyketide synthase gene type II and a ubiquitous defense enzyme phospholipase A2 in the host sponge Dendrilla nigra. Curr Microbiol 58(5):459–463

    Article  PubMed  CAS  Google Scholar 

  • Shröder HC, Brandt D, Schloßmacher U, Wang X, Tahir MN, Tremel W, Belikov SI, Muller WEG (2007) Enzymatic production of biosilica glass using enzymes from sponges: basic aspects and application in nanobiotechnology (material sciences and medicine). Naturwissenschaften 94:339–359

    Article  Google Scholar 

  • Sipkema D, van Wielink R, van Lammeren AAM, Tramper J, Osinga R, Wijffels RH (2003) Primmorphs from seven marine sponges: formation and structure. J Biotechnol 100(2):127–139

    Article  PubMed  CAS  Google Scholar 

  • Sipkema D, Franssen MCR, Osinga R, Tramper J, Wijffels RH (2005a) Marine sponges as a pharmacy. Mar Biotechnol 7:142–162

    Article  PubMed  CAS  Google Scholar 

  • Sipkema D, Osinga R, Schatton W, Mendola D, Tramper J, Wijffels RH (2005b) Large-scale production of pharmaceuticals by marine sponges: sea, cell or synthesis? Biotechnol Bioeng 90:202–222

    Article  Google Scholar 

  • Valisano L, Bavestrello G, Giovine M, Cerrano C (2006a) Primmorphs formation dynamics: a screening among Mediterranean sponges. Mar Biol 149(5):1037–1046

    Article  Google Scholar 

  • Valisano L, Bavestrello G, Giovine M, Arillo A, Cerrano C (2006b) Seasonal production of primmorphs from the marine sponge Petrosia ficiformis (Poiret, 1789) and new culturing approaches. J Exp Mar Biol Ecol 337:171–177

    Article  Google Scholar 

  • Valisano L, Bavestrello G, Giovine M, Arillo A, Cerrano C (2007) Effect of iron and dissolved silica on primmorphs of Petrosia ficiformis (Poiret, 1789). Chem Ecol 23:233–241

    Article  CAS  Google Scholar 

  • Valisano L, Pozzolini M, Cerrano C, Giovine M (2012) Biosilica deposition in the marine sponge Petrosia ficiformis (Poiret 1987): the model of primmorph reveals time dependence of spiculogenesis. Hydrobiologia 687(1):259–273

    Article  CAS  Google Scholar 

  • Vilanova E, Coutinho C, Maia G, Mourão PAS (2010) Sulfated polysaccharides from marine sponges: conspicuous distribution among different cell types and involvement on formation of in vitro cell aggregates. Cell Tissue Res 340:523–531

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Schröder HC, Brandt D, Wiens M, Lieberwirth I, Glasser G, Schloßmacher U, Wang S, Müller WEG (2011) Sponge biosilica formation involves syneresis following polycondensation in vivo. Chem Biol Chem 12(15):2316–2324

    Article  CAS  Google Scholar 

  • Zhang X, Cao X, Zhang W, Yu X, Jin M (2003a) Primmorphs from Archaeocytes-dominant cell population of the sponge Hymeniacidon perleve: improved cell proliferation and spiculogenesis. Biotechnol Bioeng 84(5):583–590

    Article  PubMed  CAS  Google Scholar 

  • Zhang W, Zhang X, Cao X, Xu J, Zhao Q, Yu X, Jin M, Deng M (2003b) Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley). J Biotechnol 100(2):161–168

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was partially supported by Italian Research Ministry Fund (PRIN 2007) to MG.

Author information

Authors and Affiliations

  1. DIMES, University of Genoa, Genoa, Italy

    Francesca Mussino & Umberto Benatti

  2. DISTAV, University of Genova, Genoa, Italy

    Marina Pozzolini, Laura Valisano & Marco Giovine

  3. DISVA, Università Politecnica delle Marche, Ancona, Italy

    Carlo Cerrano

  4. Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy

    Umberto Benatti & Marco Giovine

  5. Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università degli Studi di Genova, Corso Europa 26, 16132, Genoa, Italy

    Marco Giovine

Authors
  1. Francesca Mussino
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marina Pozzolini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laura Valisano
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carlo Cerrano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Umberto Benatti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marco Giovine
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marco Giovine.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mussino, F., Pozzolini, M., Valisano, L. et al. Primmorphs Cryopreservation: A New Method for Long-Time Storage of Sponge Cells. Mar Biotechnol 15, 357–367 (2013). https://doi.org/10.1007/s10126-012-9490-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-012-9490-z

Keywords

Search

Navigation