Isolation of Mouse Endometrial Epithelial and Stromal Cells for <em>In Vitro</em> Decidualization

Abstract

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Hyaluronic Acid Hydrogel Integrated with Mesenchymal Stem Cell‐Secretome to Treat Endometrial Injury in a Rat Model of Asherman's Syndrome

“…Human umbilical vein endothelial cells were also obtained from ATCC (CRL1730) and cultured in vascular cell basal medium (ATCC; PCS 100‐030) infused with endothelial cell growth kit‐ VEGF (ATCC; PCS 100‐041). Endometrial epithelial cells and stromal cells were separated from 3 eight‐week‐old rats according to a video protocol . The rat endometrial stromal cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) with l ‐glutamine, phenol red, 4‐(2‐Hydroxyethyl) piperazine‐1‐ethanesulfonic acid, 100 µg mL −1 gentamicin, 0.5 µg mL −1 amphotericin B, N ‐(2‐Hydroxyethyl)piperazine‐ N ′‐(2‐ethanesulfonic acid) (HEPES), and fetal bovine serum (FBS; 10% by volume).…”

Lats1 and Lats2 are required for the maintenance of multipotency in the Müllerian duct mesenchyme

“…Female Lats1 flox/flox ; Lats2 flox/flox mice ranging from 8 to 12 weeks of age were injected daily with estradiol (Sigma-Aldrich) (100 ng, subcutaneously) for 3 days prior to endometrial stromal and epithelial cell isolation and culture. All steps of cell purification, culture and in vitro decidualization were performed essentially as described by De Clercq et al (2017). Freshly isolated stromal cells were incubated overnight to permit attachment to the cell culture plates, and subsequently infected with Ad-Cre or Ad-eGFP (Vector development laboratory, Baylor College of Medicine, Houston, TX, USA) at a multiplicity of infection of approximately 50 for 24 h. Following washes, stromal cells we co-cultured with epithelial cells grown in ThinCert cell culture inserts (Greiner Bio-One International) such that the stromal and epithelial cells remained separate but were able to communicate via a semipermeable membrane.…”

Overactivated sonic hedgehog signaling aggravates intrauterine adhesion via inhibiting autophagy in endometrial stromal cells

“…Primary mouse endometrial stromal cells were obtained from three mice/group according to the protocol from JoVE 72 , and total RNA was extracted from primary endometrial stromal cells within 6 h of cell culture to be similar to the in vivo state.…”