Optimization of Extracellular Matrix for Endometrial Triple-Cell-Type Organoids Culture and Its Regenerative Potential

Abstract:\nThe success of bioengineered implants depends on the regeneration of the vascular system. To optimize the extracellular matrix (ECM) for the culture of endometrial triple-cell-type organoids, we incorporated fibrin and matrigel into a hydrogel and assessed the formation of vascular networks. We found that increasing the proportion of fibrin in the hydrogel enhanced vascular network formation until a fibrin proportion of 50% was reached. At a fibrin proportion of 70% and higher, the endometrial epithelial organoids (EEOs) exhibited significant adhesion to the culture dish. The stiffness and pore size of the hydrogel scaffold were also measured, with the hybrid F5M5 hydrogel found to provide an optimal physical environment for organoid growth and vascular network establishment. Dynamic 3D culture of the triple-cell-type endometrial organoids in a microfluidic chip resulted in the formation of a highly interconnected network and complex cellular interactions. Transcriptome sequencing revealed a striking similarity between the cultured organoids and in vivo endometrial tissue. Furthermore, the organoids responded to sex hormones, exhibiting changes in gene expression associated with decidualization and the menstrual cycle. In a mouse model, the endometrial organoids demonstrated regenerative potential, leading to thicker endometrium and greater development of endometrial glands compared to controls. These findings demonstrate the optimization of the ECM for endometrial organoid culture and highlight the potential of these organoids for endometrial regeneration.