Novel methods to establish whole-body primary cell cultures for the cnidarians Nematostella vectensis and Pocillopora damicornis

Cnidarians are emerging model organisms for cell and molecular biology research. However, successful cell culture development has been challenging due to incomplete tissue dissociation and contamination. In this report, we developed and tested several different methodologies to culture primary cells from all tissues of two species of Cnidaria: Nematostella vectensis and Pocillopora damicornis. In over 170 replicated cell cultures, we demonstrate that physical dissociation was the most successful method for viable and diverse N. vectensis cells while antibiotic-assisted dissociation was most successful for viable and diverse P. damicornis cells. We also demonstrate that a rigorous antibiotic pretreatment results in less initial contamination in cell cultures. Primary cultures of both species averaged 12–13 days of viability, showed proliferation, and maintained high cell diversity including cnidocytes, nematosomes, putative gastrodermal, and epidermal cells. Overall, this work will contribute a needed tool for furthering functional cell biology experiments in Cnidaria.

www.nature.com/scientificreports/ studies and produce reliable cell cultures that can be used as a versatile tool for live cell assays. We developed an extensive antimicrobial pre-treatment protocol to reduce microbial contamination. We tested several different medias and modified a previously developed recipe which we found was the best media to promote cnidarian cell survivability while also subduing microbe growth 31 . Using this modified media, we then monitored 123 N. vectensis cell cultures and 51 P. damicornis cell cultures. We show in both N. vectensis and P. damicornis that diverse cell types can reliably survive ex vivo for over 12 days. We also show that by using these dissociation methods, antibiotic pretreatments, and modified media that both N. vectensis and P. damicornis cell cultures have early proliferation, a high cell diversity, low rates of early microbial contamination, and consistent cell morphologies throughout the time of culturing. With these new methods, live cell methods can be developed more readily enabling the development of functional assays to better understand cnidarian cell biology.

Methods
Pre-culture animal preparation. N. vectensis. Adult N. vectensis were maintained in glass bowls containing 0.2 μm filtered 11ppt saltwater in the dark, at room temperature. Full strength saltwater was sourced from Biscayne Bay of Miami, FL, USA and diluted using reverse osmosis fresh water to bring the final concentration to 11ppt. Animals were fed 5 days per week with freshly hatched Artemia (Utah Sea). 50% water changes were done 3 times a week 33 . In preparation for cell culture, animals were removed from bowls and rinsed three times with "anemone gentamicin medium" (AGM) which consists of sterile 11 ppt saltwater supplemented with 10 μg/ml Gentamicin Reagent Solution (Gibco by Life Technologies) 31 (Table S1). For 3-7 days, individual anemones were isolated in AGM with daily media changes and starved. Each animal was then individually rinsed in a 2.5 μg/ml Penicillin/Streptomycin/Amphotericin B solution (PSAb) (Sigma) in 0.2 μm-filtered 11ppt saltwater and incubated at room temperature in 2.5 μg/ml PSAb for 10 min. Animals were then transferred into individual sterile 12-well tissue culture plates (VWR, Radnor, Pennsylvania) with selected media detailed below.
P. damicornis. Colonies of P. damicornis coral genotype, PAN-10, were originally collected from Saboga Island, Panama in 2005 and have been maintained at Rosenstiel School of Marine and Atmospheric Sciences culturing facilities 34 . The corals were maintained in an 800-gallon semi-recirculating system being constantly supplied with 10 μm-filtered sea water and were illuminated with 60 μmol·m -2 ·s -1 on a 12-h light/12-h dark cycle. The corals were fed using larval AP100 dry diet powder (Ziegler) twice per week. Coral tanks were cleaned twice per week to reduce algae. Just prior to starting the cell dissociation step, coral fragments approximately 1 cm in length were rinsed for 5 min with a transfer pipet using 0.2 μm-filtered full strength seawater. www.nature.com/scientificreports/ Tissue dissociation and plating of cell cultures. N. vectensis. We tested several different methods of dissociation including mechanical, antibiotics-facilitated with 3% PSAb, and chemical (2% N-acetyl l-cysteine and trypsin). Of these we found that mechanical dissociation generated the most viable cells ( Figure S1). For mechanical dissociation, individual anemones were treated with 7% MgCl 2 in 0.2 μm-filtered 11ppt seawater and then sliced with a scalpel into tissue clumps. In some cases, the tentacle tissue and mesenterial tissue were separated with the scalpel before dissociation. This was done to demonstrate different localized cell populations with pictures ( Fig. 3), however, the resultant cultures were not quantified in our study. These tissue clumps were then further dissociated by repeated pipetting using a wide bore 100 μl pipet tip to reduce cell damage. Following dissociation, cell slurries from one whole organism were concentrated into 1 ml and centrifuged three times at 100×g for 3 min, replacing supernatant with Leibovitz's L-15 media between each centrifugation. The resulting pellet was loosened with gentle pipetting. Then 200 μl was added to 4-5 wells of a 6-well plate with 6 ml of anemone cell culture media (ACCM) or 100 μl was added to 9-10 wells of a 12-well plate with 2.5 ml ACCM. Wells without cells were used as controls to test for media contamination. The remaining clumps were left to incubate in the media and over 24 h spontaneously dissociated to individual cells (Fig. 1A). ACCM is a modified recipe of a previously published media for N. vectensis ectodermal tissue culture 31 . ACCM consists of 80% AGM, and 20% full strength media (FSM), which is 95% L-15 Medium, 3% FBS, 1% PSAb, and 1% HEPES Buffer. 1% Penicillin-Streptomycin-Amphotericin b (PSAb) was also added to each well along with 7.5 μg/ml Plasmocin Prophylactic(InvivoGen, San Diego, California) to reduce bacteria growth. Other media were tested, but survival rates of cells were not as high as ACCM (Fig. 2E).
P. damicornis. Building on previous methods for dissociation of coral embryos, antibiotic-facilitated dissociation was used to cause expulsion of coral tissue from the adult skeleton 32 . P. damicornis fragments were submerged in 5-10 ml of antibiotic solution (enough to completely submerge the fragment): 2% PSAb, 7.5 μg/ ml Plasmocin Prophylactic, and 30 μg/ml Gentamicin in 0.2 μm-filtered seawater in 6-well cell culture plates. The fragments incubated in this solution for 24-48 h to prevent microorganism growth and to promote the expulsion of the coral tissue from the coral skeleton (Fig. 1D). This treatment caused the sloughing off of live cells and "polyp bail-out", which has been previously described in P. damicornis as a response to hypersalinity and toxicants [35][36][37] . Cells were confirmed to be viable with a 10% Trypan Blue (Gibco) exclusion test. Following antibiotic-facilitated dissociation, cells and the remaining skeletal fragments were plated in coral cell culture media (CCCM) that consisted of 20% FSM, 80% full strength 0.2 μm-filtered seawater with 10 μg/ml Gentamicin (CGM). After 5 days, the skeleton fragments were removed from culture wells with sterilized forceps. Cultures were further supplemented with 1% PSAb and 7.5 μg/ml Plasmocin Prophylactic. www.nature.com/scientificreports/ Cell culture maintenance and viability. After 1 week, media and antibiotics were changed for N. vectensis and P. damicornis cell cultures. Following this, media and antibiotics were changed every 10 days. Cultures were observed under a microscope daily for the first week and twice a week for the remaining time of the experiment. A cell culture was deemed not viable if (1) any microbial proliferation was occurring, excluding Symbiodiniaceae, (2) if fewer than ~ 100,000 living cells remained, or (3) if more than 5% of the total cells were necrotized or fragmented. Microbial proliferation was determined visually (Fig S3). We recorded when and why each cell culture lost viability, and recognized contaminants were also recorded ( Fig. 2A,B, S2).

Cell counts and viability test.
Cell culture viability and cell number were recorded starting at 5 h post cell culture establishment. A 1 mL sample of cells was pipetted out of a culture and into a 1.5 mL Eppendorf tube. The contents were then centrifuged at 100×g for 3 min and the supernatant was replaced with a working concentration of Trypan blue stain (0.4%, Gibco Life Technologies). The stained cells were then transferred to a Reichert 0.1 mm deep improved Neubauer hemocytometer and total unstained cells were counted. These counts were performed in triplicate 3 times per week for the first two weeks in both species.   Fig. 2A,B). The overall viability of the cultures was heavily dependent on the effectiveness of the tissue dissociation method. We hypothesize that the high initial cell diversity in primary cultures promotes longer cell culture viability. We define cell diversity as the number of morphologically distinct cells observed in the viable culture. However, little is understood about whether different tissue dissociation methods promote the isolation of specific populations more readily. We found that chemical dissociation was unsuccessful in N. vectensis, but that mechanical dissociation yielded clumps of viable diverse cell types that spontaneously dissociated into sheets of individual cells (Figure S1, Fig. 1A-C). Also, for the first time we showed that adult tissue of P. damicornis is effectively dissociated using an antibiotic solution (Fig. 1D-F). The antibiotic solution caused tissue to slough off of the skeleton and after 24 h yielded a diversity of cell types (Fig. 1D-F). The process of antibiotic-facilitated dissociation for coral cell culture is a convenient innovation due to microbial mitigation and production of diverse live cells from the intact skeleton occurring concomitantly. Dissociation methods for cnidarian cell culture have been challenging due to issues with extracellular matrix strength, large amounts of mucus, and the fragility of the cells 38,39 . Chemical dissociation methods including trypsin and N-acetylcysteine yielded fewer viable cells, and fewer cell types due to incomplete dissociation ( Figure S1). The development of the dissociation methods here are a promising breakthrough for cnidarian cell culture because they yield diverse cell types that successfully adapt to the cell culture environment quickly. Primary cell cultures from N. vectensis and P. damicornis are viable for 12 days on average. Of the cell culture replicates produced, 54% of N. vectensis and 55% of P. damicornis cell cultures remained uncontaminated and maintained diverse cell types for > 10 days ( Fig. 2A,B). On average, N. vectensis cell cultures survived for 12.3 days, while P. damicornis cell cultures averaged 12.7 days. This represents the first time that dissociated cells from combined tissues of a sea anemone or coral have been able to survive in cell culture for over 12 days without contamination. Here we used high replication and well-defined standards for viability, which we believe translates directly into "usability" for future live cell methods. Most previous studies worked with specific or intact tissues, or cultured cells in media with notable contamination. Among those studies, the highest definitive survival of individual cells derived from whole-body tissue appeared to be a maximum of 10 days 24 . The relatively low amount of early contamination in our cultures may be attributed to the importance of adding antibiotics at higher concentrations, as well as, the extensive rinsing and pre-treatment with antibiotics before dissociation. We also hypothesize that diluting the media to 20% allowed the cnidarian cells to outcompete any present microbes for longer periods of time. This is notable when compared to other non-diluted media tested where rapid contamination lowered viability time (Fig. 2C,D). Early contaminations were still observed on occasion, however survival up to 30 days in 3 P. damicornis cultures and 28 days in 3 N. vectensis cultures were observed ( Fig. 2A,B). Why there is such variation in the sustainability of these cultures is still not understood, but differences in individual animal microbial communities could be one reason for this, however this hypothesis would need to be tested 40,41 .
Cell counts and microscope observations were used to determine if cultures were proliferating and cell diversity was employed as an indicator of cell culture viability. From 2 days post-dissociation, cell counts of both species showed prominent proliferation, followed by a viable cell suspension that was maintained for the remaining 12 days (Fig. 2E,F). Over a 14-day period, a higher amount of cell viability was observed for cells cultured in ACCM or CCCM than cells cultured in 0.2 μm-filtered seawater with antibiotics (Fig. 2E,F). Based on these findings, we believe that ACCM and CCCM are positively affecting cnidarian cell growth and survival, to a much better extent than seawater only or other established media (Fig. 2C,D).
Cell culture cessation was primarily due to overgrowth of Thraustochytrids, a known group of cnidarian associated Stramenopiles and historical cell culture "saboteurs" 16,42 . The principal limitation to long term cnidarian cell culture success is the overgrowth of microbes, and antimicrobial methods should be done as to mitigate contamination, without affecting host cell diversity. Causes of cell culture viability loss are catalogued in Figures S2 and S3. General cell culture progression from start to finish in both species is summarized in Figure S4.
A high diversity of viable cells was observed in both cultures. Using our cell culture methods, a high diversity of cells was achieved based on complete dissociation of all tissue types (Fig. 1A-F). In N. vectensis, specific cells or structures such as cnidocytes or nematosomes (mobile multicellular structures that contains cnidocytes and are unique to the genus Nematostella), could be readily identified from whole-body cultures (Fig. 1A). To further demonstrate how cells of various tissue sources could be cultured with these methods, cultures with only mesenteries and cultures with only oral/tentacle tissue were created. Putative digestive cells were isolated in cultures using only tissue from the mesenteries and small cnidocytes and epidermal cells were isolated from tentacle and oral tissue. (Fig. 3B,C). Each of these cell types were also observed in whole-body cultures and survived up to 28 days (Fig. 3D). Nematosomes generally remained mobile in culture for up to 5 days until they dissociated into almost entirely cnidocytes, revealing much about their cellular structure.
Within P. damicornis cell cultures, immediately following dissociation, cnidocytes and Symbiodiniaceaecontaining cells were the most recognizable cell types (Fig. 1E), however diverse round and granular cell types were also abundant even up to 30 days post-dissociation (Fig. 3E). After 5 days post-dissociation, P. damicornis cell cultures showed a high cell diversity due to complete cell dissociation from the skeleton (Fig. 3E,F). The presence of diverse cell types, proliferation, and cell motility in both N. vectensis and P. damicornis is indicative of a viable cell culture that will be useful for short term functional assays. Proliferative N. vectensis cell culture 7 days pd with diverse cells of various shapes and sizes, including "pointed" round cells (white arrowhead) and globular cells, mostly unidentifiable based on morphology. (E) P. damicornis cell culture 21 days pd with low zooxanthellae counts, giving an observable variety of mostly unidentified host cnidarian cells in culture such as abundant rounded cells (arrowhead) (F) Diverse P. damicornis culture 25 days pd with smaller putative digestive round cells (arrowhead). (G) P. damicornis culture 28 days pd with 3 types of viable cnidocytes (arrows) (along with a piece of coral aragonite in frame) (H) P. damicornis culture 6 days pd with a large discharged cnidocyte (arrow). www.nature.com/scientificreports/