Publikationsdatenbank

Establishing a 3D endometrial co-culture model of early implantation with prospective application also in reproductive toxicology testing (2019)

Art

Poster

Autoren

Peter, Dominique (WE 11)

Erickson, N. A. (WE 11)

Mundhenk, L (WE 12)

Michel, Geert

Na, Ellen

Bienert, Michaela

Buck, Volker

Wittler, Lars

Thoene-Reineke, C (WE 11)

Forschungsprojekt

Save – IVF; Entwicklung eines 3D in-vitro Fertilisationszellkulturmodell in der Maus

Kongress

European Congress of Toxicologic Pathology of the EST

Köln, Germany, 17. – 20.09.2019

Quelle

Sprache

Englisch

Kontakt

Institut für Tierschutz, Tierverhalten und Versuchstierkunde

Königsweg 67
14163 Berlin
+49 30 838 61146
tierschutz@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Establishing a 3D endometrial co-culture model of early implantation with prospective application also in reproductive toxicology testing

Introduction:
In reproductive toxicology testing, animal studies are not fully replaceable due to the complexity of the reproductive cycle, the embryonic implantation and development. The combination of an endometrial co-culture with a trophoblast body generated in vitro or an ex vivo blastocyst may at least partially mirror early implantation. Such an in vitro system could be connected upstream to the in vivo studies as an initial testing platform to assess the effect of agents on the early embryo-maternal-interface.

Materials and Methods:
Endometrial epithelial and stromal cells were separately isolated from uteri of Hsd:ICR(CD-1) mice 3.5 days after plug detection by multiple digestion and filtration steps. Stromal cells were seeded in various concentrations of GrowDex® or Matrigel® in inserts followed by apical seeding of epithelial cells and the 3D culture was incubated for up to six days. The preservation of the matrix structure was analyzed via hematoxylin-eosin staining after cryo-, paraformaldehyde (PFA) or methanol-carnoy fixation. Cells were immunophenotyped using antibodies against cytokeratin AE1/AE3, Ki67, and active caspase 3 (n=4). Murine trophoblast stem cells (TS cells) were cultured with proliferation-inhibited murine embryonic fibroblasts and passaged for several weeks.

Results:
In comparison to GrowDex®, only 10.8 mg/ml Matrigel® provided best handling, stability and preservation of the gel-structure in combination with PFA-fixation. Histologically, some stromal cells formed cellular extensions. Cytokeratin-positive epithelial cells formed gland like structures, tended to aggregate apically barely forming a monolayer without high prismatic cellular morphology. Epithelial cells were partly positive for Ki67 and mostly caspase negative. TS cells formed colonies of typical, roundish, slightly raised, and clearly demarcated morphology, which compact over time.

Discussion and Conclusion:
The combination of Matrigel® with PFA-fixation yielded best results to analyze the 3D co-culture setup. The orientation and differentiation of the proliferating epithelial cells needs improvement. The generation of a trophoblast in vitro body from cultivated TS cells and its combination with the co-culture remains to be established. After establishment of this model, the system can be analyzed also for applicability in reproductive toxicology testing.