Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral,Pocillopora damicornis

Abstract

The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.

Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro

“…In vitro aragonite crystallization has been described previously in coral cell cultures of Pocillopora damicornis (11). However, ECM and SOM production was not reported, and it was later shown that P. damicornis exhibits an 80% decrease in cell viability after 7 days in culture (12).…”

“…Establishing an in vitro coral cell culture could potentially circumvent these complications and also serve as a model for studying physiological processes at a cellular level. However, no continuous coral cell lines have been developed to date, and maintenance of primary cell cultures has encountered problems such as short-term viability or contamination by unicellular eukaryotic organisms, which eventually overgrow the original coral cells (8)(9)(10)(11)(12)(13)). Here we report on the development of a culturing system that significantly facilitates studies of coral cell physiology.…”

Scleractinian coral cell proliferation is reduced in primary culture of suspended multicellular aggregates compared to polyps

“…Alternately, coral soft tissue is chemically induced to detach, in response to removal of divalent cations (Gates and Muscatine 1992;Frank et al 1994;Kopecky and Ostrander 1999;Domart-Coulon et al 2001, 2004aMass et al 2012) or exposure to a reducing agent, such as N-acetylcysteine (Peng et al 2008). Enzymatic digestion has also been widely used to dissociate the tissue into single cells (Gates and Muscatine 1992;Frank et al 1994;Helman et al 2008;Downs et al 2010).…”

“…Adherent cultures of multicellular aggregates have been used to develop models of in vitro biomineralization (Domart-Coulon et al 2001;Helman et al 2008;Mass et al 2012). Decreasing in vitro viability usually limits the timescale of experiments using such primary cultures from a few days to a few weeks, enabling only short-term study of physiological mechanisms.…”

Cell cultures from the symbiotic soft coral Sinularia flexibilis

“…Frank et al (1994) observed that cryopreserved cell cultures after approximately 1 yr were excluded by eukaryotic unicellular organisms. Again, between 1998Again, between -2004 were no successful studies published on long term cnidarian cell cultures (Frank and Rinkevich 1999;Kopecky and Ostrander 1999;Schmid et al 1999;Domart-Coulon et al 2001 even though some encouraging reports were presented (reviewed by Rinkevich 2005).…”