Hybrid Endometrial-Derived Hydrogels: Human Organoid Culture Models and In Vivo Perspectives

Abstract

The endometrium plays a vital role in fertility, providing a receptive environment for embryo implantation and development. Understanding the endometrial physiology is essential for developing new strategies to improve reproductive healthcare. Human endometrial organoids (hEOs) are emerging as powerful models for translational research and personalized medicine. However, most hEOs are cultured in a 3D microenvironment that significantly differs from the human endometrium, limiting their applicability in bioengineering. This study presents a hybrid endometrial-derived hydrogel that combines the rigidity of PuraMatrix (PM) with the natural scaffold components and interactions of a porcine decellularized endometrial extracellular matrix (EndoECM) hydrogel. This hydrogel provides outstanding support for hEO culture, enhances hEO differentiation efficiency due to its biochemical similarity with the native tissue, exhibits superior in vivo stability, and demonstrates xenogeneic biocompatibility in mice over a 2-week period. Taken together, these attributes position this hybrid endometrial-derived hydrogel as a promising biomaterial for regenerative treatments in reproductive medicine.

Keywords: bioengineering; endometrium; extracellular matrix hydrogel; organoids.

Figure 1

Rheological analyses and scanning electron microscopy of hydrogels. A) Storage modulus (strain = 1%; frequency = 1 Hz), complex viscosity (strain = 1%; frequency = 1 Hz) and oscillation stress (shear rate = 10 s⁻¹) at 37 °C for EndoECM, Matrigel, PM, PM + EndoECM (50:50) hydrogels, and other hybrid combinations [Matrigel + EndoECM (50:50), PM + EndoECM (25:75)]. The rheological properties of native porcine endometrial tissue (control) at 37 °C are also represented. There were no statistically differences between the rheological properties of PM + EndoECM hydrogel (50:50) and PM samples (black arrows) compared to those of porcine endometrial tissue. N = 3 technical replicates/group. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. Notably, PM + EndoECM hydrogel (50:50) was established as the best hybrid endometrial‐derived hydrogel and was further evaluated with subsequent analyses. B) Scanning electron microscopy images of EndoECM hydrogel, PM, and PM + EndoECM (50:50) hydrogel at 50.0 k magnification. The diameter of the collagen fibers (white arrows) was compared in EndoECM and PM + EndoECM (50:50) hydrogels; the diameter of the RADA₁₆ peptides (red arrows) was compared in PM and PM + EndoECM (50:50) hydrogels. N = 3 technical replicates/group. Scale bars = 1 µm.

Figure 2

Matrisomal protein profile in EndoECM and hybrid endometrial‐derived hydrogels. A) The protein profiles for each condition (EndoECM and hybrid endometrial‐derived hydrogels), reflecting the proportion of core matrisome and matrisome‐associated proteins. B) Venn diagram depicting the relationships of the common matrisomal components in the EndoECM and hybrid endometrial‐derived hydrogels. C) Molecular representation of the complexity of the endometrial ECM, highlighting the common matrisomal proteins identified between the EndoECM and the hybrid endometrial‐derived hydrogels. Created with BioRender.com.

Figure 3

Proteomic comparison between the hybrid endometrial‐derived hydrogel, the bovine EndoECM hydrogel, the human EndoECM hydrogel, and the native human endometrium. A) (Above) Venn diagram highlighting the relationships of the proteins identified in the three hydrogels and the native human endometrium. (Below) The significant biological processes, cellular components, and molecular functions shared between the hybrid endometrial‐derived hydrogel and the human native endometrium. B) Heatmap depicting the common (green) and different (red) biological processes between the different hydrogels and the human native endometrium. Note: Proteomics data from the bovine EndoECM hydrogel,^{[ 28 ]} human EndoECM hydrogel,^{[ 38 ]} and native human endometrium^{[ 38 ]} were retrieved from previous publications by other research groups.

Figure 3

Proteomic comparison between the hybrid endometrial‐derived hydrogel, the bovine EndoECM hydrogel, the human EndoECM hydrogel, and the native human endometrium. A) (Above) Venn diagram highlighting the relationships of the proteins identified in the three hydrogels and the native human endometrium. (Below) The significant biological processes, cellular components, and molecular functions shared between the hybrid endometrial‐derived hydrogel and the human native endometrium. B) Heatmap depicting the common (green) and different (red) biological processes between the different hydrogels and the human native endometrium. Note: Proteomics data from the bovine EndoECM hydrogel,^{[ 28 ]} human EndoECM hydrogel,^{[ 38 ]} and native human endometrium^{[ 38 ]} were retrieved from previous publications by other research groups.

Figure 4

Hybrid endometrial‐derived hydrogel supports the in vitro culture of undifferentiated hEOs and improves their differentiation to the secretory and gestational phases. A) Representative images of hEOs cultured in the hybrid endometrial‐derived hydrogel or Matrigel control captured during (from left to right) bright‐field microscopy (BF), LIVE/DEAD assay (L/D), double immunofluorescence for E‐cadherin (E‐CAD) (green) and vimentin (VIM) (red), single immunofluorescence for laminin (LAM) (red), and immunostaining for Ki67 (Ki67). Scale bars = 50 µm. B) Quantification of SPP1 secretion into the culture media of undifferentiated and differentiated hEOs grown in both hydrogels, determined by ELISA. Relative gene expression of C) SPP1, D) PAEP, E) LIF, F) 17HDSβ2, and G) SOX9 in hEOs cultured in Matrigel or hybrid endometrial‐derived hydrogel conditions determined by qRT‐PCR analysis. Data are presented as the mean fold change from N = 4 biopsies (calculated with respect to the expression in the paired Matrigel group) ± standard error of mean. *p < 0.05.

Figure 5

Evaluation of hydrogel persistence and CD68+ macrophage invasion in C57BL/6 mice 14 days after subcutaneous injections with the Endo‐NoDC, PM, and hybrid endometrial‐derived hydrogels. A) Masson's Trichrome staining of control, Endo‐NoDC, PM, and hybrid endometrial‐derived hydrogels. Aniline blue stains the collagen I fibers. B) Immunohistochemistry for CD68, which reveals a higher infiltration in PM and hybrid endometrial‐derived conditions with respect to control and Endo‐NoDC mice. Scale bar = 50 µm. D: dermis; P: panniculus carnosus; CT: connective tissue; H: hydrogel.

Figure 6

Experimental design. Characterization of the hybrid endometrial‐derived hydrogel and its applications in vitro and in vivo. Created with BioRender.com. PM: PuraMatrix; EndoECM: decellularized endometrial extracellular matrix hydrogel; hEOs: human endometrial organoids; E‐CAD: E‐cadherin; VIM: vimentin; LAM: laminin; qRT‐PCR: real‐time quantitative reverse transcription PCR; EndoECM‐NoDC: non‐decellularized endometrial extracellular matrix hydrogel.