A Natural Xenogeneic Endometrial Extracellular Matrix Hydrogel Toward Improving Current Human in vitro Models and Future in vivo Applications

Abstract

Decellularization techniques support the creation of biocompatible extracellular matrix hydrogels, providing tissue-specific environments for both in vitro cell culture and in vivo tissue regeneration. We obtained endometrium derived from porcine decellularized uteri to create endometrial extracellular matrix (EndoECM) hydrogels. After decellularization and detergent removal, we investigated the physicochemical features of the EndoECM, including gelation kinetics, ultrastructure, and proteomic profile. The matrisome showed conservation of structural and tissue-specific components with low amounts of immunoreactive molecules. EndoECM supported in vitro culture of human endometrial cells in two- and three-dimensional conditions and improved proliferation of endometrial stem cells with respect to collagen and Matrigel. Further, we developed a three-dimensional endometrium-like co-culture system of epithelial and stromal cells from different origins. Endometrial co-cultures remained viable and showed significant remodeling. Finally, EndoECM was injected subcutaneously in immunocompetent mice in a preliminary study to test a possible hypoimmunogenic reaction. Biomimetic endometrial milieus offer new strategies in reproductive techniques and endometrial repair and our findings demonstrate that EndoECM has potential for in vitro endometrial culture and as treatment for endometrial pathologies.

Keywords: 3D culture; decellularization; endometrium; extracellular matrix hydrogels; tissue engineering.

FIGURE 1

Study design. An overview of the methodology used in this study. Whole porcine uteri were decellularized to obtain acellular tissues (1, 2), the endometrium was then isolated (3) and used to create a hydrogel consisting of solely endometrial ECM (4). The physicochemical features of this hydrogel were analyzed. Different types of endometrial cells were cultured in two- and three-dimensional conditions using endometrial ECM hydrogels, collagen, or Matrigel and their proliferation was compared (5). Next, a three-dimensional endometrium-like co-culture system made of epithelial and stromal cells was developed (6). Finally, the in vivo biocompatibility of EndoECM was described in a subcutaneous murine model (7). Image created with BioRender.com.

FIGURE 2

Endometrial isolation by microdissection. (A) Schema of manual endometrial isolation. Porcine horns were cut into ring-shaped disks, opened, and cut at the luminal side of the inner circular myometrial layer to isolate the endometrium. Image created with BioRender.com. (B) Ring-shaped sections from control uterus showing uterine layers under a stereomicroscope. (C) Opened disk from control uterus during the process of microdissection under a stereomicroscope. E: endometrium, M: myometrium.

FIGURE 3

EndoECM hydrogel preparation from porcine uteri. (A) uterus before (I) and after (II) decellularization, DC endometrial tissue stock after microdissection (III), and the formed EndoECM hydrogel (IV). (B) No-DC endometrial (1, 3, 5, and 7) and DC endometrial tissue (2, 4, 6, and 8). H&E assessment of pure endometrium isolation (1, 2). Scale bars: 250 μm. Analysis of cellular material and collagen composition by trichrome staining (3, 4) and DAPI (5, 6). Immunoreactive porcine α-gal residues (brown) by DAB immunolabeling (7, 8) Scale bars: 50 μm. (C) SDS quantification after endometrial isolation, 3 or 6 washes of 30 min with mechanical agitation. ^∗P < 0.05 and ^∗∗∗P < 0.001.

FIGURE 4

DNA and protein quantification after decellularization, lyophilization, and EndoECM setup. (A) DNA quantification and fragment-size analysis in endometrial DC and No-DC wet endometrial tissue and lyophilized endometrial powder. L: ladder; 1,2,3: No-DC wet endometrial tissue replicates; 4,5,6: DC wet endometrial tissue replicates; 7,8,9: No-DC lyophilized endometrial powder replicates; and 10,11,12: DC lyophilized endometrial powder replicates. (B) Monitoring of total protein fraction, collagen, elastin, and GAGs in DC and No-DC endometrial tissue, DC and No-DC lyophilized powder, and EndoECM hydrogel. Percentages with respect to endometrial No-DC tissue or lyophilized power. Data in μg/mg. ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 5

Representative turbidimetric gelation kinetics of EndoECM hydrogels. Comparison of normalized absorbance curves and the metrics analyzed at concentration of 3, 6, and 8 mg/mL.

FIGURE 6

Ultrastructure and proteomic profile of EndoECM hydrogels. (A) Scanning electron microscopy images of EndoECM hydrogels in comparison with No-DC Endo and MyoECM hydrogels. Arrows point to remains of cellular components in No-DC Endo. Images at 30.0 k (above) and 5.00 k (below) magnifications. Scale bars are 1.00 μm and 10.0 μm, respectively. (B) Quantitative proteomic analysis of EndoECM in comparison with No-DC Endo and MyoECM by LC-MS/MS.

FIGURE 7

MTS assay of endometrial cells in two- and three-dimensional cell culture. (A) Cell proliferation in two-dimensional coating in untreated (NoTT), collagen, Matrigel, or EndoECM coated conditions. (B) Cell proliferation in 2.5D culture in collagen, Matrigel, or EndoECM hydrogels at 3 mg/mL. (C) Cell proliferation in 3D culture in collagen, Matrigel, and EndoECM hydrogels at concentration of 3 mg/mL. Two extra EndoECM concentrations, 6 and 8 mg/mL, were also tested. Statistical analysis with respect to EndoECM 3 mg/mL. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

FIGURE 8

Macroscopic remodeling of in vitro endometrium-like culture systems. (A) Experimental design. Three-dimensional endometrial-like co-cultures were constructed with primary cells (EEC-ESC constructs) or stem cell lines (ICE6-7 constructs) using EndoECM. Two seeding approaches of EECs, method A or B, were performed to include epithelial and stromal fractions. Picture created with BioRender.com. (B) Macroscopic monitoring of ICE6-7 and EECs-ESCs constructs up to 10 days. Scale bars: 50 mm.

FIGURE 9

Cell proliferation in long-term in vitro endometrium-like co-culture. (A) Positive (human endometrium, I) and negative (mouse brain, II) control tissue of Ki67 staining. Positive (III) and negative technical control (no primary antibody, IV) Ki67 staining in cellular constructs at long term. Scale bars: 150 μm. (B) Percentage of Ki67 positive cells in ICE6-7 and EECs-ESCs constructs using method A and B at day 10.

FIGURE 10

Microscopic remodeling of in vitro endometrium-like culture systems. (A) Morphological analysis of ICE6-7 and EECs-ESCs constructs at day 10 via MT staining and epithelial (E-cadherin) and stromal (vimentin) markers. Images at 40x and 100x magnifications. Scale bars are 50 μm and 5 μm, respectively. (B) Controls of morphological analysis: acellular EndoECM hydrogels and endometrium. Scale bars: 50 μm.

FIGURE 11

In vivo gelation and cytocompatibility of EndoECM hydrogels. (A) EndoECM hydrogels after 48 h of subcutaneous injection. (B) EndoECM (I, II) and No-DC Endo (III, IV) hydrogels 48 h after subcutaneous injection via MT staining. Dotted lines represent the edge between hydrogels and subcutaneous tissue. H: hydrogel; P: panniculus carnosus. F: subcutaneous fat. Scale bars are 250 μm (I, III) and 150 μm (II, IV). (C) Infiltrated inflammatory cells in EndoECM and No-DC Endo hydrogels after 48 h. (D) Percentage of infiltrated CD68 positive cells in EndoECM and No-DC Endo hydrogels after 48 h. ^∗P < 0.05 and ^∗∗∗P < 0.001.