title: Three-dimensional culture of buffalo granulosa cells in hanging drop mimics the preovulatory follicle stage.
abstract: Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three-dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly-([2-hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF-1 for 144 hr. The expression profile of nine GC-specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D-spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment.
authors.affiliation: Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
authors.forename: Monica
authors.initials: M
authors.lastname: Yadav
authors.affiliation: Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Agrawal
authors.affiliation: Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
authors.forename: Mamta
authors.initials: M
authors.lastname: Pandey
authors.affiliation: Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
authors.forename: Dheer
authors.initials: D
authors.lastname: Singh
authors.affiliation: Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
authors.forename: Suneel K
authors.identifier: http://orcid.org/0000-0002-2834-314X
authors.initials: SK
authors.lastname: Onteru
mesh_terms: D000818:Animals; D001141:Aromatase / Q000096:biosynthesis* / Q000235:genetics; D002020:Buffaloes; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008689:Methacrylates / Q000494:pharmacology; D018874:Spheroids, Cellular / Q000378:metabolism*
chemical_list: D008689:Methacrylates; D007334:Insulin-Like Growth Factor I; C005044:hydroxyethyl methacrylate; D005640:Follicle Stimulating Hormone; D001141:Aromatase; C561410:CYP19A1 protein, human
keywords: 3D culture; buffalo; granulosa cells; hanging drop; polyHEMA