Design of a Gel-in-Scaffold Hybrid Substrate for a Multicellular Endometrial Model

Infertility is a growing global health concern. A responsive endometrium provides an environment for blastocyst implantation that can determine pregnancy success. Several in vitro endometrial models have been developed using scaffolds or hydrogels to study the interactions between the endometrium and blastocysts. However, these models fail to fully replicate cellular complexity, and lack key structural features vital for successful blastocyst implantation. This thesis explores a range of cell culture substrates, including freeze-dried collagen scaffolds (S), collagen gel (CG), peptide gel (PG), collagen gel-in-scaffold (GIS-C), and peptide gel-in-scaffold (GIS-P) systems. S can be crosslinked (XL) with EDC/NHS to improve their mechanical strength, and 100-XL denotes a standard condition using a molar ratio of 5:2:1 for EDC: NHS: COO- (Col). The GIS hybrid substrates combine freeze-dried collagen scaffolds with interpenetrating hydrogels, aiming to support cell organisation, enable greater cellular complexity, and improve structural stability during long-term cell culture. Freeze-dried collagen scaffolds (S) often exhibit inconsistent structures both within and between fabrication batches due to the stochastic nature of ice nucleation during the slurry freezing process. The application of ultrasound was found to enable the nucleation temperature of collagen slurries to be precisely controlled. Compared with scaffolds formed under random nucleation at the same nucleation temperature, this approach resulted in scaffolds with a smaller and more uniform pore size while maintaining an overall level of anisotropy and interconnectivity. The ultrasound-induced nucleation provides a feasible method for achieving greater control over scaffold architecture. The GIS system was created by infiltrating S with either a CG or a PG. The resulting GIS hybrid substrates exhibited significantly higher compressive moduli than the individual components of pure gel and pure scaffold. Notably, the bulk mechanical properties of the GIS hybrid were predominantly influenced by the scaffold XL level rather than the concentration of composition of the gel component. Human dermal fibroblasts (HDFs) were cultured within different substrates to assess their spatial distribution, morphology and interactions with the surrounding matrix. Infiltrating CG into a scaffold (at least 10-XL) significantly reduced the cell-induced matrix contraction observed in CG substrates. Cell morphologies varied across culture conditions: fibroblasts in the CG substrates showed a contractile shape, those in the S substrates exhibited a stellate shape and aligned with scaffold struts, while cells in the GIS-C system remained within the gel and displayed a round morphology. Notably, HDFs in the GIS-P substrate were more elongated and exhibited a stronger affinity to the scaffold struts than those in the GIS-C system. Overall, in the GIS system, cell behaviour was primarily influenced by seeding density and gel type, while factors such as gel concentration, scaffold crosslinking, and cell loading protocol had relatively minor effects. To investigate the potential of introducing vascularisation into the system, the infiltration behaviour of human dermal microvascular endothelial cells (HDMECs) was compared across different substrate systems with pre-seeded HDFs. In CG substrates, significant gel shrinkage was observed, along with limited HDMEC infiltration and no observable vessel formation. In S substrates, HDMECs directly infiltrated the structure, and an early-stage HDMEC alignment was observed in samples supplemented with 2 ng/mL VEGF on Day 14. In GIS substrates, HDMEC infiltration was enhanced by both pre-loading HDFs and supplementing VEGF into the system. Additionally, lowering the gel concentration promoted infiltration. However, no definitive vessel formation was observed in the GIS system under any tested conditions. Finally, for endometrial modelling, a study was carried out in collaboration with the Loke Centre for Trophoblast Research, University of Cambridge. The selected GIS substrates were seeded with stromal cells and epithelial organoid fragments, cultured under two distinct conditions (standard culture and air-liquid interface approach) and compared with CG and S systems. Differences in cell proliferation and spatial organisation were observed across substrates and culture methods. Overall, cells cultured in the GIS system using an air-liquid interface method developed the most physiologically relevant endometrial structure, forming a well-defined sheet of the luminal epithelium with glands penetrating into the underlying stroma. The gel-in-scaffold hybrid system developed in this study presents a promising approach for guiding endometrial cell organisation, laying the foundation for constructing an in vitro model that more accurately represents the structural features of the human endometrium and serves as a platform for implantation studies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Song, Xinyuan

Advisor dc:contributor.advisor

• Cameron, ruth

• scaffolds
• 3D tissue model
• endometrium
• hydrogel

Licence

• https://www.repository.cam.ac.uk/bitstreams/e28b8a8b-8e45-4f8d-868d-19a33d6e0403/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.121889

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/390285