Simple SummaryDuring the early stages of embryonic development, the yolk sac (YS) performs a crucial role in performing hematopoietic, metabolic, and nutritional functions. However, the mechanism of YS transportation and its malfunction leading to yearly miscarriage is not yet clearly understood. In order to address these issues, three-dimensional (3D) culture models of the YS were created and characterized for three different domestic species: canine, bovine, and porcine. Through the utilization of specific culture media, 3D cultures were successfully generated for all three species. Afterwards, the morphology, protein, and mRNA expression related to YS functions were compared. This development represents a significant advancement as it sets the foundation for further investigation of the functional mechanisms of YS tissue.AbstractThe role of the yolk sac (YS) in miscarriage is not yet clear, largely due to ethical reasons that make in vivo studies difficult to conduct. However, 3D cultures could provide a solution to this problem by enabling cells to be arranged in a way that more closely mimics the structure of the YS as it exists in vivo. In this study, three domestic species (porcine, canine, and bovine) were chosen as models to standardize 3D culture techniques for the YS. Two techniques of 3D culture were chosen: the Matrigel®and Hanging-Drop techniques, and the 2D culture technique was used as a standardized method. The formed structures were initially characterized using scanning electron microscopy (SEM), immunohistochemistry (IHC), and quantitative real-time PCR (RT-qPCR). In general, the 3D culture samples showed better organization of the YS cells compared to 2D cultures. The formed structures from both 3D methods assemble the mesothelial layer of YS tissue. Regarding the IHC assay, all in vitro models were able to express zinc and cholesterol transport markers, although only 3D culture techniques were able to generate structures with different markers pattern, indicating a cell differentiation process when compared to 2D cultures. Regarding mRNA expression, the 3D models had a greater gene expression pattern on the Hemoglobin subunit zeta-like (HBZ) gene related to the YS tissue, although no significant expression was found in Alpha-fetoprotein (AFP), indicating a lack of endodermal differentiation in our 3D model. With the initial technique and characterization established, the next step is to maintain the cultures and characterize the diversity of cell populations, stemness, functions, and genetic stability of each 3D in vitro model.