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

Abstract

Model systems approaches search for commonality in patterns underlying biological diversity and complexity led by common evolutionary paths. The success of the approach does not rest on the species chosen but on the scalability of the model and methods used to develop the model and engage research. Fine-tuning approaches to improve coral cell cultures will provide a robust platform for studying symbiosis breakdown, the calcification mechanism and its disruption, protein interactions, micronutrient transport/exchange, and the toxicity of nanoparticles, among other key biological aspects, with the added advantage of minimizing the ethical conundrum of repeated testing on ecologically threatened organisms. The work presented here aimed to lay the foundation towards development of effective methods to sort and culture reef-building coral cells with the ultimate goal of obtaining immortal cell lines for the study of bleaching, disease and toxicity at the cellular and polyp levels. To achieve this objective, the team conducted a thorough review and tested the available methods (i.e. cell dissociation, isolation, sorting, attachment and proliferation). The most effective and reproducible techniques were combined to consolidate culture methods and generate uncontaminated coral cell cultures for ~7 days (10 days maximum). The tests were conducted on scleractinian corals Pocillopora acuta of the same genotype to harmonize results and reduce variation linked to genetic diversity. The development of cell separation and identification methods in conjunction with further investigations into coral cell-type specific metabolic requirements will allow us to tailor growth media for optimized monocultures as a tool for studying essential reef-building coral traits such as symbiosis, wound healing and calcification at multiple scales.

Fig 1. Cell yields according to dissociation method.

Average algae cell (left) and coral cell (right) yields as a factor of dissociation method (washing, soft/hard brushing, mechanical scraping, or calcium-magnesium-free seawater incubation for 1 to 24 hours) and enzyme digestion (Trypsin or Liberase), S1 Table. [ANOVA single factor: Algae df = 9, F = 10.33, p = 4.17E-10; Coral df = 9, F = 180.78, p = 3.37E-46].

Fig 2. Overall cell viability (%) according to dissociation method.

Percent viability (± SE) of cells dissociated from coral skeleton (immediately after dissociation) using different methods (soft/hard brushing, mechanical scraping, calcium-magnesium free seawater incubation for 1 to 24 hours, and enzyme digestion). S3 Table [ANOVA single factor: df = 8, F = 13.78, p = 8.85E-07].

Fig 3. Ca²⁺-Mg²⁺ free seawater incubation.

Time series photographs of Ca²⁺-Mg²⁺ free seawater coral nubbin incubation, T0 (a), 1H (b), 2H (c), 3H (d) and 24H (e). Photographs taken under a dissecting microscope.

Fig 4. Cell dissociation method combination efficacy.

Average algae cell and coral host cell yields as a factor of the origin of cell counted: Initial cell dissociation performed by scraping, remaining cells (on the skeleton) were dissociated using trypsin incubation for 1 hour. Significant difference between algae cell yield and coral cell yield by scraping and by scraping associated to trypsin digestion (scraped yield ANOVA df = 1, F = 29.84, p = 4.3E-06; scraped + trypsin yield ANOVA df = 1, F = 77.45, p = 1.57E-07) but no statistically different yields of algae cells and coral cells between methods (algae cell yield ANOVA df = 1, F = 0.057, p = 0.81; coral cell yield ANOVA df = 1, F = 0.48, p = 0.49) S3 Table.

Fig 5. Cell attachment.

Average cell attachment (%, miscellaneous Pocillopora acuta cells) on glass and tissue culture-treated (TCT) plastic, with and without collagen coating after 24H incubation at 25°C and 12 h light / 12 h dark cycle, S1 Table. (Initial cell dissociation: Ca^2+-Mg²⁺ free seawater incubation for 1 hour; Culture medium combination use: 15% DMEM, 5% FBS, 1% Penicillin-Streptomycin, 79% sterile filtered artificial seawater, see S5 Table). Significant difference between coated and uncoated substrate (df = 1, F = 6.34, p = 0.036) but no significant difference between TCT plastic and glass (df = 1, F = 2.64, p = 0.143) S5 Table.

Fig 6. Cell culture contamination variations.

Average contamination free cell culture duration (days ± SE) as a factor of media type (F12, RPMI, DMEM), serum (0%, 5%, 10%) and antibiotic (Gentamicin, Anti-Anti: Antibiotic-Antimycotic, Anti-Anti + Gentamicin, Pen-Strep: Penicillin-Streptomycin). Thirty-six combinations [media + serum + antibiotic] tested with three replicates per combination. [ANCOVA: df = 6, F = 0.48, p = 0.82].

Fig 7. Cell viability (%) of 7-day cultures.

Average overall cell viability (%, ± SE) of 7-day cultures. The cells were initially dissociated using Ca-Mg free seawater incubation for 1 hour and the culture medium used was composed of 15% DMEM, 10% FBS, 1% Penicillin-Streptomycin and 74% filtered artificial sterile seawater (replenished on days 2 and 5), n = 3. [ANOVA single factor, df = 3, F = 13.61, p = 1.65E-03].

Fig 8. Coral cell variety (microscope photographs).

Different cell types observed after dissociation from P. acuta nubbbins, coral host cells of different sizes (a, red arrow heads), single Symbiodinaceae cell (b), two symbiotic cells encapsulated inside a coral host cell (c). (d) unsorted cells composed of miscellaneous coral host cells (red arrow heads), symbiotic Symbiodinaceae (black arrow heads), deployed trichous haploneme nematocyst (yellow arrow head) and deployed mastigophore nematocyst (yellow arrow head with red outline), closed capsule of trichous haploneme nematocyst (e), spirocyst nematocyst (f) and amoebocyte-like cell (g). Photographs (a)–(c) were taken using a Cytation3 imaging plate reader with GFP and Texas Red filters. Photographs (d–g) were taken under a compound microscope (no staining).