Optimization of Extracellular Matrix for Culturing Vascularized Endometrial Triple-Cell-Type Organoids

Abstract:\n\nThe regeneration of blood vessels is critical for the success of bioengineered implants. In this study, we aimed to optimize the extracellular matrix (ECM) for the culture of endometrial triple-cell-type organoids. We incorporated human umbilical vein endothelial cells (HUVECs), endometrial stromal cells (ESCs), and endometrial epithelial organoids (EEOs) into a hydrogel containing fibrin and matrigel. We observed that the vascular network formation by HUVECs and the adhesion of EEOs were enhanced with increasing proportions of fibrin in the hydrogel. The stiffness and pore size of the hydrogel scaffold were also measured, and a hybrid hydrogel with intermediate properties was identified as optimal for organoid growth and vascular network establishment. We then constructed triple-cell-type endometrial organoids in a microfluidic chip and found that a medium with a 5:5 ratio of ECM and ExM supported the development of vascular networks and minimized EEOs adhesion. The dynamic 3D cultured organoids displayed a capillary network-like structure and complex cellular interactions. Transcriptome sequencing revealed that the gene expression pattern of the organoids closely resembled that of in vivo endometrial tissues. Furthermore, we demonstrated that the organoids responded to sex hormones and exhibited regenerative potential in a mouse model of endometrial damage. In conclusion, our optimized ECM and microfluidic chip culture system provide a valuable platform for studying endometrial regeneration and can potentially be used for tissue engineering applications.