Isolation and characterization of endometrial luminal epithelial and stromal cells in vitro

Abstract

In this study, we have described a simple method of isolating endometrial luminal epithelial (LE) and stromal (ST) cells using ovine uteri samples obtained from the abattoir. Endometrial lining were digested in trypsin, collagenase and bovine serum albumin dissolved in Hanks balanced solution. The cells were seeded into a 24-well plate at a concentration of 5 × 10⁵ per well, while some were snap frozen and kept at -800C for RNA extraction at a later date to determine the presence of leucocytes in the cell culture system. Some cells were also cultured into a 4-well chamber slide for standard immunocytochemistry. At 100% confluence, the media were removed and cells were processed for RNA extraction. The extracted RNA was used in a conventional PCR to detect the presence of progesterone and oestrogen receptors. The cell population was determined by cell morphology and immunocytochemical staining with cell specific staining of cytokeratin and vimentin. The mean yield was 49 × 10⁶ cells per 50 gm of digested endometrial tissue. Separated stromal cells were of higher purity percentage (ST; 90 ± 4%; against LE; 10 ± 4%) than epithelial cells which tended to have more stroma (LE; 83 ± 10%, against ST; 17 ± 10%). At 100% confluence (usually day 7-8), the mean percentages LE and ST in the mixed culture were 59% and 41% respectively. The LE expressed cytokeratin protein while, ST expressed vimentin markers. The absence of CD45 marker confirmed the absence of leucocyte in the cell culture system. Progesterone and oestrogen receptor were present in the cell culture. Finally, this study offers a simple method using a single digestion process to isolate mix population of endometrial LE and ST, while comparing favourable with other methods with double digestion system in term of cells yield and characteristic features of the isolated cells.

Keywords: Cytokeratin, Luminal epithelia, Oestrogen, Progesterone, Stroma, Vimentin