Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis

Abstract

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

Applying model approaches in non-model systems: A review and case study on coral cell culture

“…Methods for assessing cell membrane integrity include dye exclusion assays [e.g. trypan blue, 73 , 95 ], Evan’s blue [ 176 , 177 ], propidium iodide [ 75 ], SYTOX green [ 85 ] and enzyme release assays (e.g. lactate dehydrogenase, LDH, [ 178 ].…”

The Essentials of Marine Biotechnology

“…Consequently, standard conditions do not always support fungal or bacterial production of interesting secondary metabolites (Reich and Labes, 2017). To increase the availability of microbial biomass and minimize the environmental damage impacts of organismal collections, new strategies are needed to develop sustainable in vitro approaches for the cultivation of whole organisms or of individual cell types from targeted marine species (Rinkevich, 1999(Rinkevich, , 2005(Rinkevich, , 2011Barnay-Verdier et al, 2013;Ventura et al, 2018;Maristem COST Action 12 ). This is also of importance for sponges and corals, which host endosymbiotic intracellular microorganisms.…”

Transcription Profiling of CulturedAcropora digitiferaAdult Cells Reveals the Existence of Ancestral Genome Regulatory Modules Underlying Pluripotency and Cell Differentiation in Cnidaria

“…In cnidaria, attempts to establish permanent cnidarian cell lines have failed due to decreasing in vitro viability and proliferation (review in Rinkevich 2011 ). Despite this, short-term primary cultures have been used to understand the cellular mechanisms underlying fundamental cnidarian processes, such as symbiosis ( Barnay-Verdier et al 2013 ), calcification ( Domart-Coulon et al 2001 ; Mass et al 2012 ), thermal stress ( Nesa and Hidaka 2009 ), and regeneration ( Schmid and Alder 1984 ). These studies reported inconsistent results regarding survival, proliferation, and viability, reflecting the lack of standardized protocols (reviewed by Rinkevich 2005 , 2011 ).…”