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Abstract
To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.
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1 Chulalongkorn University, Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, CU-Animal Fertility Research Unit, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Veterinary Clinical Stem Cells and Bioengineering Research Unit, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875)
2 Chulalongkorn University, Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, CU-Animal Fertility Research Unit, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875)
3 Utrecht University, Department of Clinical Sciences, Utrecht, The Netherlands (GRID:grid.5477.1) (ISNI:0000 0000 9637 0671)
4 Chulalongkorn University, Division of Allergy and Clinical Immunology, Department of Medicine, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Faculty of Medicine, Center of Excellence in Vaccine Research and Development (Chula Vaccine Research Center-Chula VRC), Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Thai Pediatric Gastroenterology, Hepatology and Immunology (TPGHAI) Research Unit, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875)
5 Kasetsart University, Department of Physiology, Faculty of Veterinary Medicine, Bangkok, Thailand (GRID:grid.9723.f) (ISNI:0000 0001 0944 049X)
6 Chulalongkorn University, Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Faculty of Veterinary Science, Veterinary Systems Pharmacology Center (VSPC), Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875)
7 Chulalongkorn University, Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Faculty of Veterinary Science, Veterinary Systems Pharmacology Center (VSPC), Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Department of Pharmacology, Faculty of Veterinary Science, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875); Chulalongkorn University, Faculty of Dentistry, Center of Excellence in Regenerative Dentistry, Bangkok, Thailand (GRID:grid.7922.e) (ISNI:0000 0001 0244 7875)