Influence of different isolation techniques on quantity and purity of epithelial cells from the equine oviduct

Abstract

Conventional in vitro fertilization with equine gametes is not successful. In vivo, fertilization takes place in the ampulla of the oviduct. Setting up an in vitro oviduct culture system that mimics the microenvironment that gametes experience in vivo, might improve success rates of IVF. In a first step towards such a culture system, techniques for isolating epithelial cells from the ampulla of the equine oviduct were compared. Cells from oviduct segments (5 cm, slaughterhouse material) were isolated by either 1) flushing with phosphate-buffered saline, 2) longitudinal opening of the segment and mechanical isolation (scraping), or 3) filling segments with 0.5% Trypsin/0.2% EDTA solution and incubating for 30 min at 38°C before squeezing cells out (n=5 for each group). Isolated cells were diluted in Medium 199 supplemented with 20% fetal bovine serum before examining cell concentration (hemocytometer) and viability (Trypan blue staining). Flushing resulted in fewer viable cells (4.8 ± 5.5 x103; P<0.05) than scraping (25.4 ± 22.0 x106) or Trypsin digestion (31.7 ± 16.6 x106). Isolated cells were cultured for 10-14 days to form monolayers on glass coverslips. The majority of cells had a cobble-stone like appearance, typical of epithelial cells. Immune fluorescent staining of putative epithelial cells revealed the presence of vimentin in virtually all cells. At the same time, most cells stained positively for cytokeratin 19, but not for cytokeratins 4,5,6,8,10,13, or 18. In cryosections of equine oviduct (n=3) , cytokeratin 19 expression, but also cytokeratin 4,5,6,8,10,13 or 18 expression was exclusive for epithelial cells, while the vimentin was only detected in the underlying stroma cells In conclusion, epithelial cells can be successfully isolated from the equine oviduct by different methods, but de-differentiate in conventional culture conditions. Cytokeratin 19 can be used as a reliable indicator of epithelial origin.