A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

Autor: Shang P; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Stepicheva NA; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Liu H; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Chowdhury O; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Franks J; Department of Cell Biology and Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA., Sun M; Department of Cell Biology and Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA., Hose S; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Ghosh S; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Yazdankhah M; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Strizhakova A; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA., Stolz DB; Department of Cell Biology and Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA., Zigler JS Jr; Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA., Sinha D; Department of Ophthalmology, Children's Hospital of University of Pittsburgh School of Medicine, One Children's Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2021 Nov 05; Vol. 22 (21). Date of Electronic Publication: 2021 Nov 05.
DOI: 10.3390/ijms222111979
Abstrakt: Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ . These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.
Databáze: MEDLINE
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