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

• Published in: International journal of molecular sciences 2021-11, Vol.22 (21), p.11979
• Main Authors: Shang, Peng, Stepicheva, Nadezda A, Liu, Haitao, Chowdhury, Olivia, Franks, Jonathan, Sun, Ming, Hose, Stacey, Ghosh, Sayan, Yazdankhah, Meysam, Strizhakova, Anastasia, Stolz, Donna Beer, Zigler, Jr, J Samuel, Sinha, Debasish
• Format: Article
• Language: English
• Subjects: adenoviral transduction; Adenoviridae - genetics; Adenoviruses; Animals; Apoptosis; Autophagy; Biological activity; Biology; Cell culture; Cells, Cultured; Efficiency; Epithelium; explant culture; Explants; Genetic Vectors - administration & dosage; Inflammation; Inflammatory response; Kinases; Macular degeneration; Macular Degeneration - genetics; Macular Degeneration - metabolism; Macular Degeneration - pathology; Mice; Mice, Inbred C57BL; Models, Animal; Organ Culture Techniques - methods; Phagocytosis; Pharmacology; Pluripotency; Protein expression; Proteins; retina pigment epithelium; Retinal pigment epithelium; Retinal Pigment Epithelium - cytology; Retinal Pigment Epithelium - metabolism; RPE flatmount; Signal transduction; Stem cells; Transduction, Genetic; Transgenes; Tyrosine
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222111979

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 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 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 . 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.