Generation of a novel human 3d endometrium-like culture system model

BALAÑA ME; GANIEWICH D; MERESMAN G; OLIVARES C; LEIROS G; KUSINSKY G

Leiden

Congreso; 10th Edition of Leiden International (Bio)Medical Student Conference (LIMSC); 2017

INTRODUCTIONThere is a need for in vitro complex systems to study physiologic and physiopathological aspects of human endometrial biology. In particular, 3D culture systems could be important tools to advance in knowledge of the mechanisms involved in the development and progression of endometriosis. The major aim of this study was to develop an in vitro 3D culture system, imitating the architecture and physiology of the human endometrium, which could then allow examination of paracrine cross-talk between epithelial and stromal cells. This model will further be used to understanding the pathogenesis of endometriosis and could be applicable to novel drug discovery. MATERIALS AND METHODSIn this study, telomerase immortalized human endometrial stromal cells (t-HESC) and ECC-1, an established epithelial cell line derived from an adenocarcinoma of human endometrial lining, were used. 3D endometrial cell system was created by seeding T-HESC into bovine type I collagen as scaffold. After gelation, ECC-1 cells were added and the system was incubated for 3 or 7 days with RPMI with 5% fetal bovine serum (3D System 1). Another assay was performed by generation of small spheroids with stromal cells, transferred into a collagen coated plate and cultured for 96 hs. Then, epithelial cells were added to the spheroid cell culture system and allowed to incubate for another 72 hs (3D System 2). At the end of incubations, all the systems were formalin fixed and paraffin embedded and stained with haematoxylin and eosin (H-E). Immunohistochemistry assay was performed to evaluate cytokeratin expression in epithelial cells. RESULTSIn the 3D System 1, t-HESC incorporated into the type I collagen scaffold, elongated and spread-out. Clear contraction of collagen by human t-HESC cells was observed as result of its proliferation. The endometrial-like 3D architecture stained with H-E, was evaluated histologically under light microscopy. We could observe a typical stratified-like layer of epithelial cells, some of them attempted to penetrate the stromal matrix, probably indicating the beginning of spontaneous formation of early glands. No differences were found comparing 3 days with 7 days-incubated 3D cell systems, suggesting that 72 hs is enough time to generate appropriate cell culture conditions. In the spheroid cell culture we could observe a discrete migration from the spheroid to the scaffold, but worsened architecture preservation in comparison with 3D System 1.Epithelial cell origin and location were confirmed by cytokeratin positive immunostaining,CONCLUSIONA novel model of a complete endometrial 3D culture system was performed. CollagenType I scaffold provided an adequate environment for the growth of cells. Our first studies indicated that 72 hours-incubated 3D cell culture using stromal cells in suspension rather than in spheroids is simpler and more effective way to achieve the proposed objective. Gland formation appears to be stimulated, although no typical epithelial gland structure was seen in our experimental models. Further studies should be done with human endometrial primary cell cultures to improve the success in generating in vitro human endometrial like structures.