P-523 3D Sw71 blastocyst-like model and its validation by primary trophoblasts
Authors: Manchorova D , Alexandrova M , Terzieva-Karaivanova A , You Y , Dimotrova V , Mor G , Dimova T 
Published: June 2023

Abstract

Study question
Are the in vitro developed 3D-Sw71 blastocyst-like model and the primary trophoblast 3D model interchangeable?

Summary answer
We confirmed that 3D-Sw71 model can be successfully replaced by 3D primary trophoblast model.

What is known already
Overcoming the challenges and ethical considerations of manipulating human embryos, researchers use cell lines to resemble blastocyst morphology and behavior. We have constructed 3D-Sw71 blastocyst-like model from non-cancerous trophoblast cell line. Like human blastocyst, our Sw71 spheroids undergo compactization and cavitation, have similar shape and size and attach to endometrial epithelium, invade extracellular matrix and migrate between endometrial stromal cells. Models’Sw71 cells have the hybrid epithelial-mesenchymal phenotype of the extravillous trophoblasts (EVT), important for building functional placenta. 3D-Sw71 models have the unique invariant HLA-profile of EVTs (HLA-G+, HLA-C+) providing tolerogenic maternal-embryo immune recognition. Sw71-2D and 3D -cultures produced CD63-positive exosomes.

Study design, size, duration
To validate our models in vivo, we performed direct comparison between 3D Sw71 spheroids and analogous spheroids built from isolated primary chorion-derived trophoblasts (1st trimester of pregnancy). The comparison was done in context of the following criteria: generation/ differentiation, morphology (shape, size), function, invariant HLA profile, presence of hybrid phenotype and exosomes secretion. For each condition of validation at least 3 independent experiments were conducted.

Participants/materials, setting, methods
3D-Sw71 spheroids were constructed from Sw71 cells and 3D primary trophoblasts spheroids – from trophoblasts from placenta of pregnant women, directed to elective early pregnancy termination. Trophoblast cells were isolated by enzymes treatment and Percoll gradient separation. The generation and migration of the spheroids was monitored by live cells imaging and EMT and HLA molecules were detected by FACS and immunocytochemistry. The exosomes isolated by ultrafiltration were characterized by TEM, dot-blot analysis and immunogold staining.

Main results and the role of chance
From pure trophoblast cultures (&gt; 80%, assessed by FACS) were generated long-lived clones, cells from which were used for 3D primary trophoblasts spheroids construction. Our results showed that in both 3D culture settings we obtained stable, round shaped, multilayered and relatively symmetric spheroid structures followed by compaction (12–24 h) and formation of single differentiated spheroid (24-48 h) with an intact periphery and easy to manipulate. The primary trophoblasts 3D spheroids resemble 3D blastocyst-like structures in shape and size. Primary trophoblast spheroids migrated successfully in the same time frame as 3D in vitro blastocyst-like structure (BLS), (48 hours). The trophoblasts cells of the primary 3D spheroids have hybrid phenotype (Vim+Ck7+) as Sw71 cells of 3D-Sw71 BLS. These expressed the invariant HLA-G and -C molecules as well. The HLA molecules were proven also in situ in early human placenta by IHC. Sw71 and primary trophoblasts secrete exosomes, proven by TEM as 20-120 nm EV, positive for CD63 (exclusive exosome marker). In conclusion, 3D-Sw71 blastocyst-like structure represent a valuable model for in vitro trophoblast and implantation studies as well as trophoblasts-immune cells interactions.

Limitations, reasons for caution
Primary trophoblasts cultures have limited viability, which makes difficulty to perform more than three independent experiments for all conditions. In general, manually transferring 3D spheroids to new media conditions is very laborious, time-consuming and not amenable to high-throughput screenings.

Wider implications of the findings
Mimicking the implanting embryo has huge implications for a plethora of studies, such as toxicity and drug screens, development of implantation-promoting compounds. 3D-Sw71 model has a potential to integrate additional components such as immune cells, endothelial cells/vessels and bioengineered in vitro models of the uterine wall/glands to uncover human implantation.

Trial registration number
not applicable