De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering
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...
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Nature Portfolio
2024-04-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-59471-z |
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| author | Sawita Santiviparat Theerawat Swangchan-Uthai Tom A. E. Stout Supranee Buranapraditkun Piyathip Setthawong Teeanutree Taephatthanasagon Watchareewan Rodprasert Chenphop Sawangmake Theerawat Tharasanit |
| author_facet | Sawita Santiviparat Theerawat Swangchan-Uthai Tom A. E. Stout Supranee Buranapraditkun Piyathip Setthawong Teeanutree Taephatthanasagon Watchareewan Rodprasert Chenphop Sawangmake Theerawat Tharasanit |
| author_sort | Sawita Santiviparat |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T07:16:58Z |
| format | Article |
| id | doaj.art-ff2ccc1a43ed4b44a33282f53ac0284f |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-24T07:16:58Z |
| publishDate | 2024-04-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj.art-ff2ccc1a43ed4b44a33282f53ac0284f2024-04-21T11:16:18ZengNature PortfolioScientific Reports2045-23222024-04-0114111510.1038/s41598-024-59471-zDe novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineeringSawita Santiviparat0Theerawat Swangchan-Uthai1Tom A. E. Stout2Supranee Buranapraditkun3Piyathip Setthawong4Teeanutree Taephatthanasagon5Watchareewan Rodprasert6Chenphop Sawangmake7Theerawat Tharasanit8Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn UniversityDepartment of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn UniversityDepartment of Clinical Sciences, Utrecht UniversityDivision of Allergy and Clinical Immunology, Department of Medicine, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Chulalongkorn UniversityDepartment of Physiology, Faculty of Veterinary Medicine, Kasetsart UniversityVeterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Chulalongkorn UniversityVeterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Chulalongkorn UniversityVeterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Chulalongkorn UniversityDepartment of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn UniversityAbstract 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.https://doi.org/10.1038/s41598-024-59471-zEquineEndometriumROCK inhibitorThree-dimensional culture |
| spellingShingle | Sawita Santiviparat Theerawat Swangchan-Uthai Tom A. E. Stout Supranee Buranapraditkun Piyathip Setthawong Teeanutree Taephatthanasagon Watchareewan Rodprasert Chenphop Sawangmake Theerawat Tharasanit De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering Scientific Reports Equine Endometrium ROCK inhibitor Three-dimensional culture |
| title | De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering |
| title_full | De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering |
| title_fullStr | De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering |
| title_full_unstemmed | De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering |
| title_short | De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering |
| title_sort | de novo reconstruction of a functional in vivo like equine endometrium using collagen based tissue engineering |
| topic | Equine Endometrium ROCK inhibitor Three-dimensional culture |
| url | https://doi.org/10.1038/s41598-024-59471-z |
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