Background: Embryo implantation is a complex process regulated by interactions between endometrial epithelial and stromal cells. The endometrium plays a critical role in this process, providing a supportive environment for embryo attachment. However, conventional 2D cell culture models fail to fully replicate the complex 3D structure and cellular interactions of the endometrium. To overcome these limitations, 3D organoid models have been developed to better mimic the in vivo endometrial environment. Methods: In this study, a multicellular uterine organoid model was developed using porcine endometrial epithelial cells (pEECs) and porcine endometrial stromal cells (pESCs) to evaluate the effects of the endometrial environment on embryo implantation. First, single-cell endometrial organoids (pEOs) were formed by culturing pEECs in Matrigel, and their basic cellular characteristics were assessed. Then, a multicellular uterine organoid model was established by combining pEOs with pESCs. Finally, porcine embryos were co-cultured with this model to examine its effect on embryo attachment. Results: The multicellular uterine organoid model facilitated embryo attachment, demonstrating that the 3D structure and cellular interactions of the endometrium play a significant role in embryo implantation. The presence of both epithelial and stromal cells contributed to a more physiologically relevant environment that supported embryo adhesion. Conclusions: This study demonstrates that a multicellular uterine organoid model can serve as a useful in vitro system for porcine embryo implantation research. This model may contribute to a better understanding of embryo development and implantation mechanisms, with potential applications in regenerative medicine and biotechnology.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Ethical statement
    Chemicals
    Preparation of endometrial cell line
    Establishment of monocellular endometrial organoids(mono-EOs)
    Immunofluorescence
    Establishment of multicellular endometrial organoids(multi-EOs)
    Preparation of porcine embryos and embryo outgrowthwith EOs
    Statistical analysis
RESULTS
    Characterization of pEEOs
    Cultivation and characterization of porcine endometrialstromal cells
    Embryo outgrowth with multi-pEOs
DISCUSSION
CONCLUSION
REFERENCES

• Ghangyoung Kim(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea)
• Seungjun Lee(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea)
• Yunju Ha(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea)
• Jongki Cho(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea) Corresponding author
• Seonggyu Bang(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Ayeong Han(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Heejae Kang(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Sanghoon Lee(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)