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Establishment and characterization of a coculture system of equine endometrial epithelial and stromal cells
Author(s) -
Lapko L,
Böttcher D,
Theuß T,
Klug J,
Schoon HA
Publication year - 2017
Publication title -
reproduction in domestic animals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.546
H-Index - 66
eISSN - 1439-0531
pISSN - 0936-6768
DOI - 10.1111/rda.12915
Subject(s) - uteroglobin , stromal cell , immunocytochemistry , endometrium , biology , cell culture , microbiology and biotechnology , in vitro , cytokeratin , receptor , steroid hormone , hormone , chemistry , endocrinology , medicine , immunology , biochemistry , immunohistochemistry , genetics , cancer research , gene
Contents To investigate the equine endometrium as close to the in vivo situation as possible, we established a coculture system for epithelial and stromal cells ( EC s/ SC s). EC s and SC s were isolated from nine endometrial tissue specimens. EC s obtained as glandular formations were cultivated on one side of the semipermeable membrane of a Millicell ® insert. After 2 days, SC s (2 × 10 4 cells/membrane) were seeded onto the other side of the same membrane. During cocultivation, the low serum containing culture medium (Theuß et al., 2010) was supplemented with different concentrations and combinations of 17β‐estradiol (2.0–3.0 pg/ml medium) and progesterone (0.5–15.0 ng/ml medium). Once the cocultures formed continuous cell layers as determined by phase‐contrast microscopy, the membranes were fixed and processed for light microscopical examination. Cytokeratin 19, steroid hormone receptors and the uterine proteins uteroglobin and calbindin D9k were detected using immunocytochemistry to determine the degree of culture purity and functional cellular differentiation. The culture purity of the EC layer averaged ≥95%. Uteroglobin and calbindin D9k were consistently expressed in EC s, while hormone receptors were predominantly absent in both cell populations. An explicit cytomorphological epithelial differentiation with formation of round‐oval to polygonal cell forms was encountered in ≤50% of all EC s and independent of supplemented steroids. Based on the findings altogether, and despite the partly absent congruence to the in situ prerequisites, we established a standardized and reproducible coculture system, which offers a basic approach for studies of physiologic and pathophysiologic issues in the mare.