Construction of a three dimensional in vitro embryo implantation research model using alginate macro porous scaffold

Tali Tavor Reem, Dept. of Pharmaceutical Engineering, Azrieli College of Engineering, Jerusalem, Israel
Dganit Stern, School Of Pharmacy, Hebrew University, Jerusalem, Israel
Reuven Reich, School Of Pharmacy, Hebrew University, Jerusalem, Israel

Implantation failure remains an unsolved obstacle in reproductive medicine and is a major cause of infertility. Only about 20% of embryos transferred to the uterus, following in vitro fertilization (IVF), lead to the birth of a healthy infant. Due to obvious ethical restrictions, there is an unmet need to establish an in vitro model that mimics the events in the uterine wall during the implantation process. The available two-dimensional models do not fully represent the events taking place at implantation.

Endometrial cell lines, RL95-2 or HEC-1A, displaying receptive and non-receptive endometrial properties, respectively, were seeded into alginate macro-porous scaffolds, prepared by a freeze-dry technique. Cell constructs were cultivated in four different media: (a) 3 weeks in Estrogen containing medium, (b) Progesterone containing (c) hormone-free medium, or (d) one week priming of Estrogen, following by two weeks of Progesterone containing medium. Cultivation under 3D conditions within macro-porous alginate scaffolds enabled long-term cultivation of the cells for at least 4 weeks. E-cadherin mRNA expression levels, evaluated by qPCR, were shown to be hormone-dependent in RL95-2 cell constructs, in contrast to HEC-1A cells, where no hormonal effect was evident. In 2-weeks old RL95-2 constructs, Estrogen treatment significantly increased E-cadherin mRNA expression, compared to other hormone treatments. E-cadherin immuno-staining of cell constructs revealed pronounced protein expression in RL95-2 cell constructs, compared to HEC-1A. JAR spheroid attachment to 3 weeks old RL95-2 culture was confirmed by H&E staining, whereas no such attachment was evident in HEC-1A. Our 3D culture models enabled long-term culture of viable endometrial cells. These cultures may serve as a research model for studying the regulatory mechanism governing implantation process and evaluation of potential novel therapeutic strategy for regulating implantation defects and restoring the ability to implant embryos in patients with repeated implantation failure (RIF).