Background: Although an understanding of the proliferation and differentiation of fish female germline stem cells (GSCs) is very important, an appropriate threedimensional (3D) research model to study them is not well established. As a part of the development of stable 3D culture system for fish female GSCs, we conducted this study to establish a 3D aggregate culture system of ovarian cells in marine medaka, Oryzias dancena. Methods: Ovarian cells were separated by Percoll density gradient centrifugation and two different cell populations were cultured in suspension to form ovarian cell aggregates to find suitable cell populations for its formation. Ovarian cell aggregates formed from different cell populations were evaluated by histology and gene expression analyses. To evaluate the media supplements, ovarian cell aggregate culture was performed under different media conditions, and the morphology, viability, size, gene expression, histology, and E2 secretion of ovarian cell aggregates were analyzed. Results: Ovarian cell aggregates were able to be formed well under specific culture conditions that used ultra-low attachment 96 well plate, complete mESM2, and the cell populations from top to 50% layers after separation of ovarian cells. Moreover, they were able to maintain minimal ovarian function such as germ cell maintenance and E2 synthesis for a short period. Conclusions: We established basic conditions for the culture of O. dancena ovarian cell aggregates. Additional efforts will be required to further optimize the culture conditions so that the ovarian cell aggregates can retain the improved ovarian functions for a longer period of time.

INTRODUCTION
MATERIALS AND METHODS
    Fish
    Ovarian cell isolation and separation
    Culture of ovarian cell aggregates
    Hematoxylin and eosin (H&E) staining
    Reverse transcription polymerase chain reaction (RTPCR)and quantitative RT-PCR (qRT-PCR)
    Measurement of viability and size of ovarian cellaggregates
    Measurement of 17β-estradiol (E2) concentration
    Statistical analysis
RESULTS
    Investigation of the optimal cell populations for ovariancell aggregate formation
    Effects of media supplements on the culture ofovarian cell aggregates
    Effects of bFGF and GDNF on nanos2 and scp3expression
    Evaluation of germ cell maintenance and E2 synthesisof ovarian cell aggregates
DISCUSSION
CONCLUSION
REFERENCES

• Jae Hoon Choi(Department of Fisheries Biology, Pukyong National University, Busan 48513, Korea)
• Seung Pyo Gong(Department of Fisheries Biology, Pukyong National University, Busan 48513, Korea, Division of Fisheries Life Science, Major in Aquaculture and Applied Life Science, Pukyong National University, Busan 48513, Korea) Corresponding author