Cnidarian Cell Cryopreservation: A Powerful Tool for Cultivation and Functional Assays

Abstract

Cnidarian primary cell cultures have a strong potential to become a universal tool to assess stress-response mechanisms at the cellular level. However, primary cell cultures are time-consuming regarding their establishment and maintenance. Cryopreservation is a commonly used approach to provide stable cell stocks for experiments, but it is yet to be established for Cnidarian cell cultures. The aim of this study was therefore to design a cryopreservation protocol for primary cell cultures of the Cnidarian Anemonia viridis, using dimethyl sulfoxide (DMSO) as a cryoprotectant, enriched or not with fetal bovine serum (FBS). We determined that DMSO 5% with 25% FBS was an efficient cryosolution, resulting in 70% of post-thaw cell survival. The success of this protocol was first confirmed by a constant post-thaw survival independently of the cell culture age (up to 45 days old) and the storage period (up to 87 days). Finally, cryopreserved cells displayed a long-term recovery with a maintenance of the primary cell culture parameters and cellular functions: formation of cell aggregates, high viability and constant cell growth, and unchanged intrinsic resistance to hyperthermal stress. These results will further bring new opportunities for the scientific community interested in molecular, cellular, and biochemical aspects of cnidarian biology.

Intrinsically High Capacity of Animal Cells From a Symbiotic Cnidarian to Deal With Pro-Oxidative Conditions

“…Independent primary cell cultures were obtained from different A. viridis individuals and maintained as described in Ventura et al (2018) and Fricano et al (2020) . Briefly, cells were cultured in the dark in a thermo-regulated incubator (POL-EKO-APARATURA, Poland) at 20.0 ± 0.5°C.…”

“…The cell viability was defined as the percentage of viable cells relative to total cells (i.e., viable and dead cells). Cell growth rate was assessed every week using the cells counts with Evans blue method, as previously described in Ventura et al (2018) and Fricano et al (2020) . The following formula was then used to calculate the weekly growth rate:…”

“…Independent primary cell cultures were obtained from different A. viridis individuals and maintained as described in Ventura et al (2018) and Fricano et al (2020). Briefly, cells were cultured in the dark in a thermo-regulated incubator (POL-EKO-APARATURA, Poland) at 20.0 ± 0.5 • C. The optimized culture medium was replaced weekly and consisted of 20% GMIM (Gibco, Carlsbad, CA, United States), 5% fetal bovine serum (FBS; PAA/GE Healthcare, Chicago, IL, United States), 1% kanamycin (100 µg/mL, Sigma-Aldrich), 1% amphotericin B (2.5 µg/mL; Interchim, Montluçon, France), 1% antibiotic antimycotic solution (Sigma-Aldrich), 1% L-glutamate (Sigma-Aldrich), and 71% of filtered ASW.…”

“…In addition, the sensitivity of A. viridis to thermal and UV stresses has been well described and some mechanisms of bleaching have been decrypted, including oxidative stress and apoptosis ( Richier et al, 2006 ; Moya et al, 2012 ). Recently, we succeeded in the establishment of primary cell cultures from A. viridis exhibiting a gastrodermal signature ( Barnay-Verdier et al, 2013 ; Ventura et al, 2018 ; Fricano et al, 2020 ). Thus, to test the hypothesis of an intrinsic resistance of animal cells to H 2 O 2 , we exposed A. viridis specimens and primary cell cultures at the same H 2 O 2 concentrations (200 and 500 μM) during the same periods of time (24 h and 7 days), and compared the respective responses.…”

Help Me, Symbionts, You’re My Only Hope: Approaches to Accelerate Our Understanding of Coral Holobiont Interactions

“…Advances in biomedicine have been facilitated by the development of immortal cell lines for molecular manipulation and microscopy analysis. The generation of cnidarian cell lines has been met with limited success (see Rosental et al 2017;Fricano et al 2020;Nowotny et al 2021), although achievements have been made with respect to in vivo symbiosis (Kawamura, Sekida, et al 2021). Functional cell lines of cnidarian models will allow researchers to apply exogenous biochemical stimuli, such as TGFβ and nitric oxide, which would expand our knowledge of gene function.…”