Optimizing Extracellular Matrix for Vascularized Endometrial Triple-Cell-Type Organoids Culture and Its Potential for Endometrial Regeneration

Abstract:\u003cbr\u003eThe regeneration of the vascular system is crucial for the success of bioengineered implants. In this study, we aimed to optimize the extracellular matrix for the culture of endometrial triple-cell-type organoids. We introduced endometrial epithelial organoids (EEOs), endometrial stromal cells (ESCs), and human umbilical vein endothelial cells (HUVECs) into a fibrin-matrigel hydrogel at different ratios. We found that the addition of fibrin to matrigel enhanced the formation of vascular networks by HUVECs, and a fibrin proportion of 70% or higher promoted adhesion of EEOs. The stiffness and pore size of the hydrogel scaffold were also measured, and a hybrid hydrogel with a fibrin-matrigel ratio of 5:5 was found to provide an optimal physical environment for organoid growth and vascular network formation. Furthermore, we established a dynamic 3D culture system using a microfluidic chip, and evaluated different media formulations for co-culturing EEOs and vasculature. The ExM/ECM (5:5) medium was found to be the optimal choice for cultivating vascularized endometrial organoids. Under dynamic flow conditions, the triple-cell-type endometrial culture complex formed a highly interconnected network encompassing organoids. Immunofluorescence staining confirmed the presence of endothelial cells, stromal cells, and epithelial cells within the organoids. The dynamic 3D cultured organoids displayed a capillary network-like structure and complex cellular interactions. High-throughput transcriptome sequencing revealed a striking similarity between the organoids and in vivo endometrial tissues in terms of gene expression patterns. Furthermore, the organoids responded to sex hormones and showed signs of endometrial regeneration in a mouse model of endometrial damage. Overall, this study provides insights into optimizing the extracellular matrix for the culture of endometrial triple-cell-type organoids and demonstrates their potential for endometrial regeneration.