Ananya Ghosal, MAKAUT (West Bengal)
The cells of Cnidarians have complex microbiomes which are difficult to eliminate with antibiotics. Hence, cells of cnidarians are difficult to culture. Cnidarians are critical for the marine ecosystem. The standard growth medium is made up of antibiotics, chemicals, and seawater which reaches the optimal formulas for different cnidarian species.
Corals are marine invertebrates who belong to a class of Cnidaria. This article reports the first-ever primary cell culture study of two species of Cnidarians: Nematostella Vectensis (a type of sea anemone) and Pocillopora damicornis (a type of coral). Physical dissociation is a successful method for diverse and viable N. vectensis cells. The primary culture of both species averaged maintained high cell diversity, viability, and proliferation.
How was this accomplished?
The methods for obtaining a primary cell culture of the corals are:
- Preculture animal preparation– N. vectensis was maintained in a glass bowl which contains 0.2µm filtered 11ppt salt water at room temperature in dark. After removing the animals from the bowl and wash it with AGM (Anemone Gentamicin Medium). Individual anemones were isolated in AGM with starved and daily media change for 3-7 days. Each animal was then rinsed in 2.5µg/ml PSAb solution individually in 0.2µm- filtered 11ppt saltwater and incubate in 2.5µg/ml PSAb solution for 2 minutes at room temperature. Individually animals were transferred into a sterile 12 well tissue culture plate with selected media.
- Damicornis was maintained in 800 gallons semi recirculating system which constantly supplies 10µm of filtered seawater illuminated with 60 μmol·m-2s-1 on a 12h light/12h dark cycle. Twice per week corals were fed with larval AP100 dry diet powder. To reduce algae tanks were cleaned twice per week. Before the cell dissociation step, coral samples of 1cm length were washed with 0.2µm filtered full-strength seawater.
- Tissue dissociation and plating– Mechanical dissociation gives rise to viable cells. Anemones were treated in 0.2µm filtered 11ppt seawater with 7% MgCl. By repeated pipetting results in dissociating of tissue clumps by using a wide bore 100µl pipette tip which reduces the chance of cell damage. Cell slurries were concentrated into 1ml and centrifuged twice for three minutes at 100×g. Between each centrifugation, the supernatant is being replaced with Lebovitz’s L-15 media. In a six-well plate, 200µm was added with 6ml of ACCM in 4-5 wells.
A control well was there to test media contamination 7.5µg/ml. Plasmocin Prophylactic and PSAb solution was added to each well. In P. damicornis antibiotic facilitated dissociation was used where fragments were submerged in an antibiotic solution of 5-10ml in six-well cell culture plates. To prevent microorganism growth and promote expulsion of coral tissue the fragments were incubated for 1-2 days. Cells were viable with a 10% Trypan Blue exclusion test. The remaining skeletal fragments were plated in coral cell culture media by using sterilized forceps skeletal fragments were removed from culture wells after 5 days.
- Maintenance and viability– Media and antibiotics were changed after 1 week and followed by 10 days. The cell culture was not viable due to microbial poly filtration, less than 100,000 living cells. 5% or more than the cell was fragmented.
- Cell counts and viability test– In a 1.5ml Eppendorf tube 1ml of the sample of the cell was pipetted out from the culture. Centrifugation at 100×g for 3 minutes and replacing the supernatant with Trypan blue. The unstained cells were counted in triplicate 3 times per week for the first two weeks for both species.
Observation:
The dissociation method affects cell culture viability. Clumps of viable diverse cell types which is dissociated in sheets of individual cells yielded by mechanical dissociation. P. damicornis is dissociated by antibiotic solution, chemical dissociation method yielded few viable cells. Primary cell culture is viable for 12 days on average. N. vectensis cell culture survived for 12.3 days whereas, P. damicornis cell culture survived for 12.7 days on average. Coral has been able to survive over 12days without contamination. High diversity of viable cells was observed based on complete dissociation based on all tissue types.
Significance of the study:
Cnidarians have not yet been very intricately studied, be it their anatomy or functional validation. This might be because of the lack of access to cnidarian cells. This study is significant because it can serve as a good specimen for a detailed study on cnidarians and corals.
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Reference:
- Nowotny, James D., et al. ‘Novel Methods to Establish Whole-Body Primary Cell Cultures for the Cnidarians Nematostella Vectensis and Pocillopora Damicornis’. Scientific Reports, vol. 11, no. 1, Feb. 2021, p. 4086. www.nature.com, doi:10.1038/s41598-021-83549-7.https://www.nature.com/articles/s41598-021-83549-7
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