Cryopreservation and Cryobanking of Cells from 100 Coral Species

When coral species become extinct, their genetic resources cannot be recovered. Coral cryobanks can be employed to preserve coral samples and thereby maintain the availability of the samples and increase their potential to be restocked. In this study, we developed a procedure to determine coral species-specific requirements for cryobank freezing through determining suitable cryoprotective agents (CPAs), CPA concentrations, equilibration times, holding durations, viability rates, and cell amounts for banked coral cells, and we established the first ever coral cell cryobank. Coral cells, including supporting and gland cells, epidermal nematocysts, Symbiodiniaceae and symbiotic endoderm cells (SEC) were found from the extracted protocol. Approximately half of the corals from the experimental corals consisted of spindle and cluster cells. Gastrodermal nematocysts were the least common. The overall concentration of Symbiodiniaceae in the coral cells was 8.6%. Freezing using DMSO as a CPA was suitable for approximately half of the corals, and for the other half of species, successful cell cryopreservation was achieved using MeOH and EG. EG and DMSO had similar suitabilities for Acanthastrea, Euphyllia, Favites, Lobophyllia, Pavona, Seriatopora, and Turbinaria, as did EG and MeOH for Acropora, Echinopyllia, and Sinularia and MeOH and DMSO for Platygyra after freezing. At least 14 straws from each species of coral were cryobanked in this study, totaling more than 1884 straws (0.5 mL) with an average concentration of 6.4 × 106 per mL. The results of this study may serve as a framework for cryobanks worldwide and contribute to the long-term conservation of coral reefs.


Introduction
Coral reefs are economically, socially, and environmentally valuable. It primarily acts as a barrier that reduces waves and protects the coasts from damage. Coral reefs act as habitats, shelters, and nurseries for various marine organisms (i.e., nudibranchs, clownfish, etc.) [1,2]. In addition, coral reefs fix nitrogen and carbon by converting harmful gases to harmless gases, which act as a filtration system in the ocean [3,4]. Additionally, it may generate revenue for nations such as Australia (The Great Barrier Reef), which draws a large number of visitors each year to witness the wonderful view of marine life [2,5]. Corals can also be used for medical purposes such as anti-inflammatory, anti-cancer, bone repair, and neurological treatment [6]. However, an increasing number of factors, such as coral bleaching, coral diseases, environmental degradation, and overfishing, have led to a coral crisis [7,8]. In addition, coral bleaching has become increasingly common with climate changes due to global warming [9,10]. By 2030, 60% of the global reef area may be lost [11]. When a species of coral becomes extinct, its genetic resources cannot be recovered [12]. Coral loss can eventually lead to broad ecological effects, especially on organisms that coexist with coral and rely on it as a source of food or shelter [13].
This coral crisis has led to the emergence of coral genetic cryobanks. At cryobanks, samples are frozen and preserved at low temperatures, ensuring the year-round availability in fresh seawater in a flow-through system tank (0.6 t) with a salinity of 33-35 ppt and a flow rate of 7500 L/h, which was achieved with a wavemaker (R35210, ReefWave, Israel). The corals were broken into chunks (4 cm 2 ) with chisel-like steel tools during collection. The collected wild corals were kept for a maximum duration of 7 days for the experiment. Cultured corals were obtained through husbandry at the NMMBA, and the minimum age of the cultured coral was 3 years. The coral collection was approved by the Kenting National Park Management Office.

Coral Identification
The corals were first identified and categorized by divers during the collection process. The categorization was confirmed at the laboratory on the basis of bone plates. The polyp and sclerite morphologies were analyzed under a light microscope (C31, Olympus, Japan), and the relevant features (e.g., corallite structure) were checked against a key to determine the species of the sample corals. The samples were maintained in a fixation buffer (10% sodium hypochlorite solution; Sigma-Aldrich, St. Louis, MO, USA) before being rinsed with distilled water and dried. The samples were subsequently transferred to a different laboratory for independent testing. The two laboratories obtained identical species-level identification results.

Coral Host Cell and Symbiodiniaceae Extraction
An extraction solution was prepared to separate cells from the corals. The solution comprised 3% w/v N-acetyl cysteine (Sigma-Aldrich) and 0.5% w/v trypsin (Sigma-Aldrich) in 25 mL of filtered seawater. In addition, 0.8% w/v NaOH (Sigma-Aldrich) was dissolved in the extraction solution to increase its pH to 8. 2-8.4. The coral solutions were then shaken at 100 rpm with an orbital shaker (MS-NRK-30, Major Science, Taiwan). Color was monitored periodically to determine the extraction progress. The cells were spun (25 • C, 2000 rpm, 3 min) using a refrigerated centrifuge (5810R, Eppendorf, Germany) to wash away the extraction solution, which was replaced with filtered seawater. A 0.63 × 32 mm 2 needle syringe (23G × 1 1 4 R.B.; Top, Japan) was used to break apart the cell chunks in the solution. The tubes (Falcon, NY, USA) were covered in aluminum foil to protect the samples from light. The coral cell types were identified based on the microscopic photography data from [48][49][50].

Cryopreservation
CPAs were prepared using filtered seawater and 1 or 2 M ethylene glycol (EG; J.T. Baker, NJ, USA), methanol (MeOH; Darmstadt, Germany), or dimethyl sulfoxide (DMSO; Sigma-Aldrich). The freezing procedures entailed adding the CPAs to the samples at a ratio of 1:3. The mixtures were equilibrated at room temperature (25 • C) for 10 or 20 min, and the equilibrated samples were loaded into 0.5-mL straws (IMV Technologies, France) and suspended above liquid nitrogen for 10 min for cooling at a rate of approximately 60 • C/min on a cooling device (Taiwan patent no. M394447). The straws were immersed in a liquid nitrogen bath for at least 30 min. The straws were subsequently thawed for 10 s in a 40 • C water bath (SWB-10L-1, Major Science, Taiwan), and 1-mL samples of coral cells treated with each combination of CPA type, CPA concentration, and equilibration time were obtained for viability testing (Figure 1). and suspended above liquid nitrogen for 10 min for cooling at a rate of approximately 60 °C/min on a cooling device (Taiwan patent no. M394447). The straws were immersed in a liquid nitrogen bath for at least 30 min. The straws were subsequently thawed for 10 s in a 40 °C water bath (SWB-10L-1, Major Science, Taiwan), and 1-mL samples of coral cells treated with each combination of CPA type, CPA concentration, and equilibration time were obtained for viability testing (Figure 1).

Viability Assay
Adenosine triphosphate (ATP) bioassays (Cellular ATP Kit HTS; BioThema, Handen, Sweden) and a hemocytometer (Neubauer-improved bright line; Marienfeld Superior, Germany) were used to test viability. An ATP viability assay can be used to determine the energy produced by cells for metabolism by using a solution that employs luciferase and D-luciferin to catalyze the release of light. For each measurement, 50 µL of sample and ATP reagent were combined in a luminometer tube and mixed for 3 min. The coral cells were then inserted into the luminometer (Lumat 9507, Berthold Technologies, Bad Wildbad, Germany) to obtain ATP readings. Cell counts were also used to assess the cell density of the samples before and after the experiments. A hemocytometer and a microscope (CX31, Olympus, Japan) were used for cell counting. The cells in a control sample were counted immediately after the extracted sample was washed, and the cells of a contrast sample were counted after the sample was thawed. A microscopy camera (518CU,

Viability Assay
Adenosine triphosphate (ATP) bioassays (Cellular ATP Kit HTS; BioThema, Handen, Sweden) and a hemocytometer (Neubauer-improved bright line; Marienfeld Superior, Germany) were used to test viability. An ATP viability assay can be used to determine the energy produced by cells for metabolism by using a solution that employs luciferase and D-luciferin to catalyze the release of light. For each measurement, 50 µL of sample and ATP reagent were combined in a luminometer tube and mixed for 3 min. The coral cells were then inserted into the luminometer (Lumat 9507, Berthold Technologies, Bad Wildbad, Germany) to obtain ATP readings. Cell counts were also used to assess the cell density of the samples before and after the experiments. A hemocytometer and a microscope (CX31, Olympus, Japan) were used for cell counting. The cells in a control sample were counted immediately after the extracted sample was washed, and the cells of a contrast sample were counted after the sample was thawed. A microscopy camera (518CU, ACCU-SCOPE, New York, NY, USA) and photo editing software (SE3 Micrometrics, Taiwan) were used with light microscope (CX31, Olympus, Japan) to photograph the coral cells under 400× magnification.

Coral Cryobanking
Coral cells were cryobanked in 0.5-mL straws (IMV Technologies, Normandy, France) using the optimal freezing conditions determined through the aforementioned assessment. Each straw was denoted by a four-digit number. The first digit indicated whether the coral was cultured or wild, the second and third digits, respectively, indicated the genus and species of the coral, and the fourth digit indicated the type of coral (e.g., massive, branching, foliaceous, encrusting, columnar, laminar, or free-living). The straws were inserted into a goblet mounted on an aluminum cane in a numbered cannister, which was later inserted into the stainless-steel canister of a cryogenic storage system (GT38 Air Liquide, Cryopal, France) for long-term storage ( Figure 2).
Coral cells were cryobanked in 0.5-mL straws (IMV Technologies, Normandy, France) using the optimal freezing conditions determined through the aforementioned assessment. Each straw was denoted by a four-digit number. The first digit indicated whether the coral was cultured or wild, the second and third digits, respectively, indicated the genus and species of the coral, and the fourth digit indicated the type of coral (e.g., massive, branching, foliaceous, encrusting, columnar, laminar, or free-living). The straws were inserted into a goblet mounted on an aluminum cane in a numbered cannister, which was later inserted into the stainless-steel canister of a cryogenic storage system (GT38 Air Liquide, Cryopal, France) for long-term storage ( Figure 2).

Statistical Analysis
Statistical analysis was performed in SPSS (version 17.0; SPSS, Illinois, USA). The one-sample Kolmogorov-Smirnov test and Levene test were used to verify the normality and homogeneity of the data. A one-way analysis of variance and Least Significant Difference's post hoc test were then performed to identify differences associated with the type of CPA, CPA concentration, and equilibration time. The data are presented as means ± standard errors of three replicates; a p of < 0.05 was considered significant.

Statistical Analysis
Statistical analysis was performed in SPSS (version 17.0; SPSS, Illinois, USA). The onesample Kolmogorov-Smirnov test and Levene test were used to verify the normality and homogeneity of the data. A one-way analysis of variance and Least Significant Difference's post hoc test were then performed to identify differences associated with the type of CPA, CPA concentration, and equilibration time. The data are presented as means ± standard errors of three replicates; a p of <0.05 was considered significant.

Coral Cell Types
The coral tissue was composed of spherical gland cells with single ( Figure 3A) or multiple vesicles ( Figure 3B), which secrete mucus. Supporting cells are the key components of the epidermis; they are host cells found in both singular ( Figure 3C) and cluster form ( Figure 3D). All cnidaria species contain epidermal nematocysts, which enable predation; in this study, they were present as microbasic p-mastigophores in the shape of a crescent ( Figure 3E), spiral ( Figure 3F), capsule ( Figure 3G), or encapsulated needle ( Figure 3H). Symbiodiniaceae ( Figure 3I) were frequently discovered in symbiotic coral. The SECs comprised single or multiple Symbiodiniaceae ( Figure 3J,K) and were harbored in a layer of host lipid bodies, which enable photosynthesis. SECs are unique in that their host lipid bodies can expand to accommodate Symbiodiniaceae ( Figure 3K). Spindle cells (cells that overlap) were found with the Symbiodiniaceae ( Figure 3L) and had a similar shape and size to those of gland cells. Cluster cells were observed aligned in their normal form ( Figure 3M). Disintegrated Symbiodiniaceae, which had dark, greenish coloration, were dispersed within the cells ( Figure 3N). Ruptured cluster cells with disintegrated Symbiodiniaceae leaking into their inner cells were also identified ( Figure 3O). Gastrodermal nematocysts (holotrichs) were only found in the gastrodermis; they appeared as tubules throughout and without a shaft ( Figure 3P). crescent ( Figure 3E), spiral ( Figure 3F), capsule ( Figure 3G), or encapsulated needle (Figure 3H). Symbiodiniaceae ( Figure 3I) were frequently discovered in symbiotic coral. The SECs comprised single or multiple Symbiodiniaceae ( Figure 3J,K) and were harbored in a layer of host lipid bodies, which enable photosynthesis. SECs are unique in that their host lipid bodies can expand to accommodate Symbiodiniaceae ( Figure 3K). Spindle cells (cells that overlap) were found with the Symbiodiniaceae ( Figure 3L) and had a similar shape and size to those of gland cells. Cluster cells were observed aligned in their normal form ( Figure 3M). Disintegrated Symbiodiniaceae, which had dark, greenish coloration, were dispersed within the cells ( Figure 3N). Ruptured cluster cells with disintegrated Symbiodiniaceae leaking into their inner cells were also identified ( Figure 3O). Gastrodermal nematocysts (holotrichs) were only found in the gastrodermis; they appeared as tubules throughout and without a shaft ( Figure 3P).
Both 10 and 20 min of equilibration time resulted in a 5-20% change in coral cell viability after freezing. The extraction duration for the coral ranged from 30 to 76 min. Cells from corals such as Anthelia glauca, Favia favus, Montipora altasepta, Montipora foliosa, Montipora aequituberculata, Pachyseris speciosa, Platygyra lamellina, and Pachyseris rugosa were not successfully extracted because slime formation led to cell clumping. Cells from 100 coral species were cryobanked, and at least 14 straws were obtained from each species, for a total of 1884 straws (0.5 mL) with an average concentration of 6.4 × 10 6 /mL.

Identification of Cell Types
The cell types identified for each coral species are presented in Table 2. High ratios of gland cells, supporting cells, epidermal nematocysts, Symbiodiniaceae, and symbiotic endoderm cells (SECs) were discovered in more than 80 coral species, indicating that basic coral cells can be easily obtained in high concentrations through the aforementioned extraction process. Among the 101 coral species, approximately half comprised spindle and cluster cells. Gastrodermal nematocysts were the type of cell identified least often. This indicates that some coral cells in deeper layers can be extracted through our protocol. Porites Porites • Availability of cells. Numbers 1-20 were corals cultured for more than 3 year at the NMMBA. * Data for Sinularia compressa unavailable.

Symbiodiniaceae Concentration in Cells of Coral Species
The percentage of Symbiodiniaceae in each coral species is presented in Figure 4. More than 90% of the species contained Symbiodiniaceae. The remaining species, including the azooxanthellate coral Tubastraea aurea, had no Symbiodiniaceae. Notably, azooxanthellate coral do not contain or thus rely on Symbiodiniaceae to survive. Symbiodiniaceae were present in the coral cells at an average concentration of 8.6%. The five corals with the most abundant Symbiodiniaceae were E. aspera, S. sadensis, S. flexibilis, E. paraancora, and P. verrucosa. The lowest concentrations of Symbiodiniaceae were found in A. subulata, Acropora nana,

Discussion
Coral gametes, tissue balls, larvae, and Symbiodiniaceae were first cryopreserved through various techniques in the 2010s [51]. However, different coral materials may be more or less compatible with various freezing techniques. The size, shape, lipid content, and chilling sensitivity of the coral as well as the CPA and ice formation of the coral may affect the success of cryopreservation. Coral sperm have been extensively preserved because of the large volume of obtainable samples; such samples can be used for seeding purposes and contain abundant genetic biomaterial [52,53]. Coral sperm cryopreservation is commonly performed through two-step freezing with DMSO [10,19,54,55]. For coral oocytes, cryopreservation with MeOH has demonstrated promising results [56,57]. However, oocyte cryopreservation with MeOH requires vitrification with EG and propylene glycol [58,59] because high concentrations of MeOH can be toxic to oocytes. Nevertheless, attempts to cryopreserve asymbiotic [60] and symbiotic [61,62] coral larvae through vitrification and laser nanowarming have been successful.
Membrane-permeating CPAs, such as DMSO, EG, and MeOH, have low molecular weights and can diffuse freely (if the equilibration time is sufficient) across membranes to protect cells from cold shock, chilling injury, and dehydration stress [64,67,68]. In this study, we discovered that no CPA could be applied to all coral species. However, DMSO, MeOH, and EG were suitable CPAs for several coral types. DMSO can strip water and metal ions [69], increase permeability by disintegrating bilayer structures [70], and prevent crystallization [71]. DMSO was effective on >50% of the corals in our experiments. DMSO is also suitable for coral sperm [10,19,72] and tissue balls [65,66]. By contrast, MeOH can prevent osmotic stress and preserve the gene expression, mitochondrial DNA, and lamina of nuclear envelopes [58,73]. In our study, MeOH was highly suitable for H. microconos (69%), M. valenciennesi (50%), and Acropora muricata (47%) after cryopreservation and was a suitable CPA for the genera Acropora and Platygyra. MeOH has also demonstrated effectiveness in the cryopreservation of the energy of coral (Echinopora sp.) oocytes [58] and the viability and fertility of coral (J. juncea and J. fragilis) sperm sacs [55] and was suitable for most Symbiodiniaceae, including Symbiodinium, Breviolum, Cladocopium, Fugacium, and Gerakladium, when used in a two-step vitrification and freezing method [14]. EG is similar to DMSO and MeOH with respect to membrane permeability and protection against fluorescence leakage [74][75][76]. In this study, EG was suitable for P. clavus, H. microconos, Favites flexuosa, and H. coerulea and achieved >50% viability after cryopreservation, indicating EG performed equally to MeOH. However, EG was less effective in protecting P. damicornis tissue balls against CPA toxicity than MeOH, glycerol, and DMSO were [65]. EG combined with other CPAs has been used in vitrification solutions for J. juncea oocytes (Tsai et al., 2015) and symbiotic coral (S. caliendrum and P. verrucosa) larvae cryopreserved through vitrification and laser nanowarming [33,61].
Many aspects of coral cell function have been studied. Coral gland cells, which are secretory cells known as mucocytes [79,80], have been reported to transform into mucous cells [81] that function as antibacterial protectors, particle traps, and energy carriers [79,82,83]. In stony corals, supporting cells secrete a calcareous skeleton [84]. Only cnidaria have epidermal and gastrodermal nematocysts [85][86][87]. Morphological observations in the present study revealed that nematocysts have crescent-shaped, spirocyst, capsule-shaped, and encapsulated needle forms representing the developmental stages of coral tentacles, a finding supported by Ostman et al. (2010) [50]. Nematocysts play an essential role in defense against predators, locomotion, and host invasion [85,88,89]. SECs, which are Symbiodiniaceae combined with endodermal cells, enable nutrients and energy to flow between corals and hosts [90]. SECs have a unique capacity for expansion to enable the housing of more Symbiodiniaceae when necessary [49,91].
In the present study, 18 of 26 types of coral cell (e.g., granular gland cells, pigment cells, supporting cells, bipolar neurons, calicoblasts, desmocytes, epitheliomuscular cells, neurons, interstitial cells, interstitial stem cells, absorptive cells, and nutritive-muscular cells) were not identified. These coral cell types can be identified through single-cell RNA sequencing, scanning electron microscopy, transmission electron microscopy, histology, and the use of cell markers [37,45,46,48,84,[92][93][94][95]. Rosental et al. (2017) [95] and Synder et al. (2020) [96] have used fluorescence-activated cell sorting to separate symbiotic and asymbiotic populations in in vitro cultures. However, this method is limited to identifying these two main populations. Single-cell RNA sequencing is the most effective method for studying cell-cell interactions and cell morphology and physiology in nonmodel species [48] and for identifying cell markers to screen for compounds indicative of coral cell functions [95].
In our study, small and round gland and supporting cells were abundant after cryopreservation. This can be attributed to their size (3-10 µm) and shape. Host coral cells are generally smaller (3-15 µm) and have a low permeability rate (1-10 µm 2 /s). Teardropshaped cells protrude and hinder diffusion because of their polarity; this does not occur in circular cells [97,98]. Some (<10%) of the Symbiodiniaceae extracted from our sampled corals were not the target cells; however, this did not affect the cellular ATP results. The 20-min equilibration time and 10-min holding time were insufficient for Symbiodiniaceae cryopreservation through the two-step freezing process. The appropriate equilibration times for the Symbiodiniaceae cryopreserved using two-step freezing with a 50 • C/min-100 • C/min cooling rate differ for different clades, such as Symbiodinium (30-60 min), Breviolum (20 min), Cladocopium (30 min), Durusdinium (30 min), Fugacium (30-60 min), and Gerakladium (30 min; Di Genio et al., 2021). Slightly longer equilibration times result from diffusion constraints caused by Symbiodiniaceae walls, both in hospite and in culture [99,100].
Various viability tests have been conducted on coral biomaterials using fluorescent metabolism markers [39], formazan spectrophotometry [43,101,102], flow cytometry with SYTOX Green [103], ATP assay [104], trypan blue exclusion testing [105], Neubauer chambers [106], fluorescein diacetate, and conventional propidium iodide [56,63]. In the present study, we used an ATP bioassay because of its ability to accurately and rapidly assess viability in only a few coral samples [11,14,33,61,71,103]. The assay detects ATP from the light emitted from the reaction of luciferin and luciferase [107,108]. Cell density was also calculated and may be an ideal proxy for determining coral health.
Mucus secretion, coral collection, and seasonal variation may have limited our experiment. Excessive mucus secretion can cause coral cells to coagulate, resulting in energy loss [79] and enhanced bacterial growth [109]. In this study, Favia speciosa, F. favus, M. foliosa, M. altasepta, P. speciosa, and Platygyra lamellina demonstrated signs of excessive mucus buildup that trapped cells, rendering impossible the separation of cells for cryopreservation. Furthermore, the collection site experiences drastic temperature variations due to a nearby power plant, strong tidally induced upwellings, and typhoons [110]. High temperatures can affect coral Symbiodiniaceae by increasing reactive oxygen species production, which can lead to oxidative stress [111,112]. An increase in sea surface temperature occurred from May to August and led to mass coral bleaching at Houbihu, Kenting. Most of the corals at the site were bleached, thus preventing coral collection from August to November, until the area demonstrated signs of recovery. Although the corals recovered, their ability to withstand cryopreservation may have been weakened because their energy was focused on recuperation after bleaching [113]. Increases in sea surface temperatures and the frequency of bleaching events may challenge the survival of coral species.
This was the first study on coral cell cryopreservation and cryobanking in which various CPAs, CPA concentrations and equilibration times were applied to numerous coral species. The cells of 100 coral species were cryobanked, with at least 14 straws for each species, for a total of 1884 straws (0.5 mL) with minimum concentrations of 1 × 10 6 /mL. To sustain the world's coral reefs, cryobanking coral cells is crucial; cryobanked cells can be used for reculture, nuclear transfer cloning, recombinant DNA, gene delivery, and genome editing. Cryobanking is a new form of coral cell preservation; the results of this study may serve as a framework for cryobanks worldwide and may contribute to the long-term conservation of coral reefs.

Conflicts of Interest:
On behalf of all authors, the corresponding author states that there is no conflict of interest.