Factors Affecting Primary Cultures of Abalone Haliotis discus hannai Ovary-dissociated Cells and General Culture Aspects

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    We investigated factors affecting primary cultures of Pacific abalone Haliotis discus hannai ovary-dissociated cells to identify general aspects of their early-phase culture. Ninety-seven cell populations derived from 30 individuals were cultured in different media with varying compositions of medium supplements, and initial attachment, subculture, and survival for ≥10 weeks were assessed according to medium composition and individual. We also examined the time required for subculture and the rate of cell death according to both culturing period and passage number within 10 weeks. A lack of fetal bovine serum (FBS) and hemolymph significantly inhibited the growth of cultured cells, while we detected no significant effect of medium composition on initial cell attachment. Through data reallocation, with the omission of data from cell populations cultured in FBS-free and hemolymph-free media, we showed that growth inhibition was also affected by individual differences among the abalones used. During the culture, we observed four different types of cell morphology. Moreover, considerable time was required for subculture—18.4 and 19.5 days for first and second subcultures, respectively—and cell death did not occur within 30 days or for passage 0. Our results will provide valuable information for developing universal cell culturing guidelines in abalone species and suggest the feasibility of culturing abalone ovary-dissociated cells.


    Haliotis discus hannai , Ovary , Primary culture , Medium supplement

  • Introduction

    Abalones which belong to mollusc are important in aquaculture, and their cell lines can be used as in vitro models for biological and pathological research on marine molluscs (van der Merwe, 2010; Pichon et al., 2013; Yoshino et al., 2013). In particular, in vitro cultures of germline stem cells such as ovarian or spermatogonial stem cells have more utility than somatic cells due to their greater developmental competence; they can be applied to the production of transgenic animals and the preservation of species through cryopreservation (Guan et al., 2006; Zou et al., 2009; Lacerda et al., 2010; Wong et al., 2013). Therefore, developing cell culture techniques for germline stem cells in abalone is important to future biotechnological applications of this organism, and thus we must establish general guidelines for abalone cell culture.

    To date, considerable effort has been put into culturing abalone cells, including hemocytes (Lebel et al., 1996; Auzoux-Bordenave et al., 2007; Farcy et al., 2007; van der Merwe et al., 2010; Gaume et al., 2012; Latire et al., 2012) and tissue-dissociated cells derived from the gills (Suwattana et al., 2010; Gaume et al., 2012; Pichon et al., 2013), heart (Suwattana et al., 2010), larvae (Naganuma et al., 1994; van der Merwe et al., 2010), mantle (Poncet et al., 2000; Sud et al., 2001; Poncet et al., 2002; Suja and Dharmaraj, 2005; Auzoux-Bordenave et al., 2007; Suja et al., 2007), and radula (Kim et al., 2014a). However, despite these efforts, culture outcomes thus far have been generally unsatisfactory. The factors involved in cell survival and growth in vitro, and the conditions required to induce stable cultures in vitro, remain unknown. Consequently, cultures can generally only be maintained for less than 84 days (Naganuma et al., 1994), aside from an exceptional case of 370 days (Suja et al., 2005).

    For these reasons, we conducted primary cultures of cell populations derived from Pacific abalone Haliotis discus hannai ovaries to determine optimal culture conditions. We determined which components of the culture medium are involved in in vitro cell survival and growth, and assessed the feasibility of culturing abalone ovary-dissociated cells by evaluating their general culture characteristics. Based on initial culture conditions from our previous reports, which showed the importance of media salinity and growth factor addition in primary cultures of radula-derived abalone cell populations (Kim et al., 2014a), we carried out primary cultures of H. discus hannai ovary-dissociated cells and evaluated the effects of media supplements. We also examined general culture characteristics such as cell morphology, time required for subculture, and cell death according to culture period and passage number.

    Materials and Methods

      >  Animals

    Pacific abalones were obtained from the Genetics & Breeding Research Center of the National Fisheries Research and Development Institute (Geoje, Korea). To obtain mature ovaries, abalones with swollen, dark green ovaries were selected and transported directly to the laboratory. The ovaries contained many late vitellogenic oocytes with distinct nuclei and nucleoli, and were classified as stage III of maturity (Najmudeen, 2007). Thirty abalones were sacrificed for this study, with an average shell length and total weight of 9.85 ± 0.36 cm and 99.07 ± 9.07 g, respectively.

      >  Hemolymph preparation

    Prior to dissecting the abalones for tissue collection, we collected hemolymph from the body cavities around the heart using 3-mL syringes as a medium supplement. Hemocytes were separated by centrifugation at 3,500 g for 15 min, and the supernatant hemolymph was kept at 4℃ overnight. The next day, after additional centrifugation at 3,500 g for 30 min, the supernatant was inactivated at 56°C for 30 min, filtered with a 0.1-μm syringe filter (Sartorius Stedim Biotech, Göttingen, Germany), and stored at –20℃ until use.

      >  Tissue dissociation and cell collection

    Abalones were disinfected in 70% ethanol (SK Chemicals, Sungnam, Korea) for 2 min, and the swollen, dark green ovaries surrounding the hepatopancreas were dissected with sterile scissors and tweezers. Two abalones were sacrificed for each replicate. Ovaries were placed in 35-mm petri dishes (SPL life Sciences, Pocheon, Korea) and rinsed five times with washing solution (WS) consisting of 0.2 μm filtered seawater supplemented with a 1% (v/v) mixed solution of penicillin and streptomycin (P/S; Gibco, Grand Island, NY, USA) and 2.5 μg/mL amphotericin B (Mediatech, Manassas, VA, USA). For cell dissociation, ovaries were finely minced with a surgical blade in an enzymatic solution consisting of Leibovitz’s L-15 medium (L15; Gibco), adjusted to 35 psu by dissolving Red Sea salt (Red Sea, Houston, TX, USA), supplemented with 0.05% trypsin–EDTA (Gibco) and 500 U/mL collagenase type I (Worthington Biochemical Corporation, Lakewood Township, NJ, USA), and incubated for 30 min at 23℃. After enzyme inactivation via the addition of L15 adjusted to 35 psu containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) P/S, all tissue derivatives were filtered on a 40-μm cell strainer (SPL Life Sciences) and centrifuged at 400 × g for 4 min. After removing the supernatant, retrieved cells were resuspended in L15 adjusted to 35 psu containing 1% (v/v) P/S. Viable cells were counted with a hemocytometer (Paul Marienfeld GmbH and Co. KG, Lauda-Königshofen, Germany) after Trypan Blue (Gibco) staining.

      >  Culture media and supplements

    Basal culture medium was L15 supplemented with 1% (v/v) P/S, 1% (v/v) nonessential amino acids (Gibco), 100 μM β-mercaptoethanol (Gibco), and 2 nM sodium selenite (Sigma-Aldrich, St. Louis, MO, USA). To enhance cell proliferation, seven supplements consisting of 15% (v/v) FBS (Gibco), 15% (v/v) hemolymph, 10 ng/mL basic fibroblast growth factor (bFGF; Gibco), 25 ng/mL epidermal growth factor (EGF; Sigma-Aldrich), 1000 U/mL leukemia inhibitory factor (LIF; Millipore, Billerica, MA, USA), 50 μg/mL abalone embryo extract (AEE), and 50 μg/mL medaka embryo extract (MEE) were added to the basal culture medium. The final salinity of all culture media was adjusted to 35 psu. MEE was prepared according to the method reported by Lee et al. (2013). Briefly, collected blastula embryos of Oryzias dancena were homogenized, processed in three freeze–thaw cycles, and centrifuged twice at 3,500 g for 30 min at 4℃ and 18,000 g for 30 min at 4℃. Of the resulting three layers, the middle layer, which contained the embryo extract, was retrieved, sterilized with a 0.1-μm syringe filter (Sartorius Stedim Biotech), and stored at ‒20℃ until use. For AEE preparation, sperm and eggs from mature H. discus hannai males and females that had been induced to spawn by sequential exposure to air and UV-irradiated seawater were artificially fertilized. When embryos reached the blastula stage, embryo extract was obtained and stored using the same method as for MEE preparation.

      >  Cell culture

    Dissociated cells were seeded in 0.1% gelatin (Sigma- Aldrich)-coated 48-well culture plates (Becton Dickinson, Franklin Lakes, NJ, USA) filled with 800 μL of culture medium at 2 × 105 cells/well density; 200 μL of mineral oil (Sigma-Aldrich) was used to cover the medium to prevent its evaporation. Cells were cultured in an 18℃ incubator in an air atmosphere. After 3 days of culture, cells were washed twice with WS and filled with fresh culture medium. Thereafter, half the media were replaced every 4 or 5 days. When cells reached 100% confluency and cell sheets were formed, we conducted subculturing. For the subculture, cells were washed twice with 1% (v/v) P/S-containing calcium magnesium-free artificial seawater (CMFAS) consisting of 26.2 g/L NaCl, 0.67 g/L KCl, 4.62 g/L Na2SO4, 0.21 g/L NaHCO3, and 0.37 g/L Na2∙EDTA and trypsinized with CMFAS containing 0.05% trypsin–EDTA. After detachment of cells, L15 adjusted to 35 psu containing 10% (v/v) FBS and 1% (v/v) P/S were added for trypsin inactivation, and the cell suspension was centrifuged at 400 g for 4 min. The collected cells were plated at different ratios according to the subculture. Until passage 2, cells were subcultured at a 1:1 ratio in a 48-well culture plate, while from passage 2 to 3, cells were split at a 1:2 ratio in a 48-well culture plate. From passage 3 to 4, cells cultured in two wells of a 48-well culture plate were subcultured together in one well of a 24-well culture plate (SPL life Sciences) and from passage 4 to 5, cells were split at a 1:2 ratio in a 24-well culture plate. In all subcultures, 50% (v/v) conditioned media were used. Initial cell attachment, survival, and cell morphology during maintenance were visually investigated under an inverted microscope (TS100-F; Nikon, Tokyo, Japan).

      >  Hoechst 33342 staining

    To investigate the state of the nuclei in cultured cells, we conducted nucleus staining. Live cells after initial attachment (passage 0) and the fourth subculture (passage 4) were washed twice with WS, and fresh media containing Hoechst 33342 (Molecular Probes, Eugene, OR, USA) at a final concentration of 5 μg/mL were added. After incubation for 30 min, cells were washed twice with WS and culture wells filled with fresh culture medium. Stained nuclei were visualized under an inverted microscope equipped with a fluorescent lamp (Nikon).

      >  Experimental design and statistical analysis

    To investigate