Poster CC2 - Engineered biomaterials for placental research

Introduction: Maternal mortality in the United States has been increasing over time and is a significant public health issue that must be addressed. In the body, pregnancy is established once an invading blastocyst implants into the endometrium, the lining of the uterus. Cell-cell crosstalk between maternal endometrial cells and fetal trophoblast cells from the blastocyst coordinate the depth of invasion into the endometrium. Too much or too little trophoblast invasion can result in a variety of pregnancy disorders and placental problems; however, implantation occurs 6-12 days after conception which renders it virtually impossible to study this process in real-time in humans. In humans, blastocyst implantation into the endometrium has never been observed and what we currently understand is inferred from rare human histological specimens. With complex, three-dimensional in vitro model systems, we have the important opportunity to study elements of this process in a controlled in vitro environment using human cells.

Existing studies in placental research utilize Matrigel, a combination of extracellular matrix proteins (e.g., laminin, entactin, collagen IV) for culturing placental cells. Importantly, significant batch-to-batch variability and inconsistent mechanical properties are major limitations to using Matrigel and render it impossible to parse how mechanical cues influence placentation. To target these limitations, we engineer biomaterials for placental research that support long-term culture of placental cells while also decoupling key material features (e.g., stiffness, viscosity). Using gelatin methacryloyl hydrogel composites combined with other natural polymers, we have created a unique suite of engineered biomaterials capable of supporting trophoblast organoids.

Materials and

Methods: As previously described (1), we synthesized and characterized a suite of gelatin methacryloyl (GelMA) hydrogels from low, medium, and high bloom strength porcine gelatin type A (Sigma). We used these GelMA variants to create composites using Cultrex and Matrigel, which are commonly used for culturing organoids and stem cells. With these biomaterial composites, we then cultured cells from the uteroplacental interface and assessed their behavior in the composites. We created spheroids from the Swan71 trophoblast cell line (ABM T0532), extravillous trophoblast organoids, and trophoblast stem cell organoids (Riken CT30) which we encapsulated within the biomaterials or cultured on top. We characterized key features for each cell type, including morphology and viability, differentiation into multiple lineages, and longevity of the cells. All methods were performed as described previously with minor modifications (1-4).

Results, Conclusions, and Discussions: Immunofluorescent staining confirmed that the trophoblast organoids are maintaining their identity when cultured in GelMA hydrogel variants (Fig. 1). Our results indicate that GelMA hydrogel variants support long-term culture of trophoblast organoids and that their differentiation into multiple lineages can be tuned based on the material properties of the hydrogel. Our optimization efforts revealed that trophoblast organoids can grow in GelMA hydrogels; however, the addition of basement membrane biomolecules (e.g., Collagen IV and Laminin) promote trophoblast invasion into the gel matrix.

In conclusion, we have engineered a suite of biomaterial composites for placental research. We demonstrate that the composite material properties influence a variety of cellular characteristics, including morphology and differentiation into multiple cell lineages. Ongoing work includes characterizing the material properties of the composites using spherical indentation to identify which material properties influence trophoblast behavior. A deeper understanding of how material properties influence cells at the uteroplacental interface will provide critical insights toward our understanding of human reproduction.

Acknowledgements (Optional): [1-4]

References and Publications:
1. Zambuto, S. G., et al., J Mech Behav Biomed Mater 2024, 154, 106509. DOI 10.1016/j.jmbbm.2024.106509.
2. Zambuto, S. G., et al., Placenta 2024. DOI 10.1016/j.placenta.2024.03.006.
3. Karvas, R. M., et al., Methods Mol Biol 2024, 2767, 85-103. DOI 10.1007/7651_2023_496.
4. Karvas, R. M., et al., Cell Stem Cell 2023, 30 (9), 1148-1165 e7. DOI 10.1016/j.stem.2023.08.005.