P-792 Human endometrial organoids embedded in hybrid endometrial-derived hydrogels as a biocompatible treatment for endometrial pathologies in preclinical models
Authors: Gómez-Álvarez M , Bueno-Fernandez C , Agustina-Hernández M , Rodríguez-Eguren A , Francés-Herrero E , Romeu M , Faus A , Pellicer A , Cervelló I 
Published: July 2024

Abstract

Study question
Are human endometrial organoids (hEOs) labelled with Molday-ION Rhodamine B (MIRB) within hybrid endometrial-derived hydrogels a biocompatible approach to treat endometrial pathologies in immunocompetent mice?

Summary answer
The direct intrauterine injection of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels demonstrated xenogeneic biocompatibility in preclinical models over a 2-week period.

What is known already
The lack of effective treatments for endometrial pathologies emphasizes the urgent need for novel bioengineering approaches. In this context, hEOs are presented as promising candidates for tissue regeneration, since they offer a native and partially undifferentiated cellular source with high proliferative capacity, which is fostered after its culture within hybrid endometrial-derived hydrogels (Gómez-Álvarez et al. 2023). Their concurrent injection aims to enhance proliferation and differentiation through its rich biochemical composition. No prior study has successfully labelled and injected hEOs in immunocompetent models, which is crucial to discern whether the therapeutic effect operates at paracrine level or involves direct tissue regeneration.

Study design, size, duration
This study was approved by an ethics committee. Decellularized porcine endometrium was used to create endometrial-derived hydrogels as previously described and then mixed with PuraMatrix to obtain the hybrid endometrial-derived hydrogel. After 7 days of culture, hEOs (passages 6-7) were labelled with MIRB for final tracking. Finally, hEOs were mixed with the hybrid hydrogel and injected into the uterine horns in immunocompetent mice (n = 3). After two weeks, biocompatibility was evaluated through blood and macroscopical analyses.

Participants/materials, setting, methods
After 7 days, hEOs were collected and labelled with MIRB (50 µg/ml) for their final tracking in the murine endometrium. Endometrial-derived hydrogel (8 mg/ml) was mixed with PuraMatrix 1% (50:50 ratio) to generate the hybrid hydrogel. After its characterization and in vitro validation (Gómez-Álvarez et al. 2023), it was used as the bioactive carrier. Each animal received a single intrauterine injection (50 µl, ∼100.000 cells). Blood, spleen, and uterus were collected after 2-week period.

Main results and the role of chance
Hybrid endometrial-derived hydrogels demonstrated biocompatibility and in vivo stability in mice over a 2-week period (Gómez-Álvarez et al. 2023). In this study, we aim to use this hybrid hydrogel with hEOs as a biocompatible treatment in preclinical models. After 7 days of culture, hEOs were successfully labelled with MIRB (∼85% efficacy), which was confirmed under fluorescence microscopy. The single intrauterine injection (50 µl, ∼100.000 cells) did not cause apparent adverse immune reactions in mice (n = 3) during the 2-week period. All mice exhibited no signs of distress and their behavior remained within normal parameters. Furthermore, the macroscopic examination of uterine horns revealed normal appearance, characterized by the absence of anomalies and tumors. At day 14 post-injection, haematocrit analyses showed normal white blood cell count: 0.89 ± 0.53 · 109/L neutrophils; 7.82 ± 2.23 · 109/L lymphocytes; 0.21 ± 0.13 · 109/L monocytes; 0.04 ± 0.04 · 109/L eosinophils; 0.00 ± 0.00 · 109/L basophils. In addition, no significant changes in red blood cells (9.31 ± 0.54 · 1012/L) and platelets (1063.00 ± 318.58 · 109/L) were observed. Histological analyses will be perform to assess the presence of hEOs labelled with MIRB in the murine endometrium and dilucidate their mechanism of action.

Limitations, reasons for caution
This study has been carried out in healthy C57BL/6 mice. To confirm the scope of this technique, the next step is to explore the regenerative potential of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels in murine models with endometrial damage.

Wider implications of the findings
These findings demonstrate the biocompatibility of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels in immunocompetent C57BL/6 mice. This bioengineering approach holds the potential to improve the treatment landscape for specific endometrial pathologies.

Trial registration number
Not applicable