Isolation and in vitro culture of primary cell populations derived from ovarian tissues of the rockfish, Sebastes schlegeli

This study was conducted to identify the general conditions for the isolation and in vitro culture of ovary-derived cells in rockfish (Sebastes schlegeli). The effects of three different enzymes on cell retrieval from ovarian tissues were evaluated first, and then the ovary-dissociated cells were cultured under various culture conditions, with varying basal media and culture temperatures, addition of growth factors, and/or culture types. We found that collagenase type I treatment was effective for cell isolation from ovarian tissues. From a total of 42 trials to evaluate the effects of basal media and culture temperatures on cell culture of ovary-dissociated cells, we observed that Leibovitz’s L15 medium was more supportive than Dulbecco’s modified Eagle’s medium for culture, and the cells could grow at all three temperatures tested, 15, 20, and 25 °C, at least up to passage 2. However, growth factor addition did not improve cell growth. Introduction of suspension culture after monolayer culture expanded the culture period significantly more than did monolayer culture alone. Our results may provide a basis for developing an in vitro system for S. schlegeli germline cell culture, which will ultimately lead to improvement of the species. Background Establishment of an in vitro system can provide a useful tool for biotechnology applications as well as various research areas (Lakra et al., 2011; Smagghe et al., 2009; Smith, 2006; Stacey, 2012). Particularly, in vitro utilization of germline cells that possess greater developmental potential can offer a novel way of deriving superior animals through grafting this in vitro system onto existing genetic breeding and transgenic technology (Gong et al., 2008; Shin et al., 2008; Kim et al., 2009; Kim and Izpisua Belmonte, 2011). In fish, however, a limited number of studies has been conducted to establish in vitro systems using germline cells, and most of those have been restricted to small fish models (Hong et al., 2004; Kawasaki et al., 2012; Nóbrega et al., 2010; Wong and Collodi, 2013; Wong et al., 2013). Thus, there has been little research regarding farmed fish species (Lacerda et al., 2010). * Correspondence: [email protected] Department of Fisheries Biology, Pukyong National University, Busan 608-737, Korea Laboratory of Cell Biotechnology, Department of Marine Biomaterials and Aquaculture, College of Fisheries Science, Pukyong National University, Busan 608-737, Korea Full list of author information is available at the end of the article © 2016 Ryu et al. Open Access This article is International License (http://creativecommons reproduction in any medium, provided you g the Creative Commons license, and indicate if (http://creativecommons.org/publicdomain/ze Rockfish (Sebastes schlegeli) is a major farmed marine finfish, representing the second largest production of fish cultured in Korea (Jayasinghe et al., 2015). Thus, attempts to develop an in vitro system for culturing germline cells followed by improvement of the breed are important due to its commercial scale as a valuable food resource. In this study, as a first step towards the longterm goal of establishing a germline cell culture system in S. schlegeli, we first conducted primary cell cultures derived from S. schlegeli ovarian tissues to establish general guidelines for S. schlegeli cell culture by determining optimal culture conditions. We first compared the effects of three different enzymes on cell retrieval from ovarian tissues for efficient cell isolation and subsequently cultured the ovary-dissociated cells under various conditions to evaluate the effects of basal media, culture temperature, growth factors, and culture type on in vitro culture.