Publikationsdatenbank

Establishment of a murine 3D cell culture model of the endometrium (2019)

Art

Poster

Autoren

Peter, Dominique (WE 11)

Erickson, Nancy (WE 11)

Mundhenk, Lars (WE 12)

Michel, Geert

Na, Ellen

Bienert, Michaela

Buck, Volker

Wittler, Lars

Thoene-Reineke, Christa (WE 11)

Forschungsprojekt

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

Kongress

22nd European Congress on Alternatives to Animal Testing/19nd Annual Congress of EUSAAT

Linz, Austria, 10. – 13.10.2019

Quelle

ALTEX proceedings

Bandzählung: 8

Heftzählung: 1

Seiten: 154

ISSN: 2194-0479

Sprache

Englisch

Verweise

URL (Volltext): https://eusaat-congress.eu/images/2019/pdf/altex_Linz2019_full.pdf

Kontakt

Institut für Tierschutz, Tierverhalten und Versuchstierkunde

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

Abstract / Zusammenfassung

In reproductive research, animal studies are not fully replaceable due to the complexity of the reproductive cycle, the embryonic implantation and development. However, in vitro models may benefit toward reduction of animals necessary. The combination of a murine endometrial co-culture comprised of stromal and epithelial cells with a trophoblast body generated in vitro or an ex vivo blastocyst may at least partially mirror early embryo implantation and facilitate the analysis of receptivity markers and early embryonal-endometrial-interaction in vitro, thereby contributing to the 3R concept.
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 using modified protocols (1-3). Based on previously published articles (1, 4), stromal cells were suspended in GrowDex® or Matrigel® of various concentrations and seeded in 24-well inserts. After gel solidification, epithelial cells were seeded onto the gel surface and the 3D cell culture was incubated basolaterally and apically with respective cell-specific medium for up to six days. Following cryo-, paraformaldehyde (PFA), or methanol-carnoy fixation, matrix structure and cellular preservation were examined macroscopically and histologically by hematoxylin-eosin (HE) staining. Cell immunophenotyping was performed via cytokeratin AE1/AE3-, Ki67- and active caspase 3-antibodies (n = 4).
For in vitro trophoblast body generation, murine trophoblast stem cells (TS cells) were cultivated on gelatin-coated MEF (mouse embryonal fibroblasts) pre-seeded plates. Cells were passaged and banked over several weeks according to established protocols (5).
Only 10.8 mg/ml Matrigel® provided acceptable handling, highest stability, as well as optimal macroscopic and, in combination with PFA-fixation, histologic preservation of the gel-structure compared to GrowDex® and lower concentrations of Matrigel®. Fixation with methanol-carnoy resulted in a loosened gel-structure whilst cryo-fixation caused difficulties in cutting with a consequently massive loss of structure. Histology revealed some stromal cells forming cellular extensions in both GrowDex® and Matrigel®. Epithelial cells, which were positive for cytokeratin, formed gland-like structures in Matrigel®, but tended to aggregate apically rather than forming a monolayer and lacked high prismatic cellular morphology. Some of the epithelial cells were positive for Ki67, whilst most of the cells were caspase negative. TS cell colonies displayed a typical roundish colony morphology with defined demarcation toward MEF cells and increasing density over time.
This modified protocol provides reproducible isolation and separation of both endometrial stromal and epithelial cells from the same organ in one process for respective mouse strain and time point of cycle. Hence, it contributes to the 3R by reducing the numbers of organs necessary compared to protocols facilitating the isolation of only one cell type at a time. In combination with 10.8 mg/ml Matrigel® and consecutive PFA-fixation, it also yielded best structural results in terms of 3D culture setup and preservation, in which co-cultured epithelial cells show active proliferation for up to six days with a low rate of apoptosis. However, epithelial cell density and morphology requires further optimization. The generation of a trophoblast in vitro body from cultivated TS cells and its combination with the co-culture remains to be established.