A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD
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 <i>in vitro</i> models t...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/21/11979 |
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| author | Peng Shang Nadezda A. Stepicheva Haitao Liu Olivia Chowdhury Jonathan Franks Ming Sun Stacey Hose Sayan Ghosh Meysam Yazdankhah Anastasia Strizhakova Donna Beer Stolz J. Samuel Zigler Debasish Sinha |
| author_facet | Peng Shang Nadezda A. Stepicheva Haitao Liu Olivia Chowdhury Jonathan Franks Ming Sun Stacey Hose Sayan Ghosh Meysam Yazdankhah Anastasia Strizhakova Donna Beer Stolz J. Samuel Zigler Debasish Sinha |
| author_sort | Peng Shang |
| collection | DOAJ |
| description | 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 <i>in vitro</i> 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 <i>in situ</i> 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 <i>in situ</i>. 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. |
| first_indexed | 2024-03-10T06:01:16Z |
| format | Article |
| id | doaj.art-fdcc07a5c43e4d50967ebb8968f7dac1 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T06:01:16Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-fdcc07a5c43e4d50967ebb8968f7dac12023-11-22T21:00:54ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-11-0122211197910.3390/ijms222111979A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMDPeng Shang0Nadezda A. Stepicheva1Haitao Liu2Olivia Chowdhury3Jonathan Franks4Ming Sun5Stacey Hose6Sayan Ghosh7Meysam Yazdankhah8Anastasia Strizhakova9Donna Beer Stolz10J. Samuel Zigler11Debasish Sinha12Department of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Cell Biology and Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USADepartment of Cell Biology and Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADepartment of Cell Biology and Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USAWilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USADepartment of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, PA 15224, USADegeneration 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 <i>in vitro</i> 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 <i>in situ</i> 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 <i>in situ</i>. 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.https://www.mdpi.com/1422-0067/22/21/11979retina pigment epitheliumexplant cultureRPE flatmountadenoviral transduction |
| spellingShingle | Peng Shang Nadezda A. Stepicheva Haitao Liu Olivia Chowdhury Jonathan Franks Ming Sun Stacey Hose Sayan Ghosh Meysam Yazdankhah Anastasia Strizhakova Donna Beer Stolz J. Samuel Zigler Debasish Sinha A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD International Journal of Molecular Sciences retina pigment epithelium explant culture RPE flatmount adenoviral transduction |
| title | A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD |
| title_full | A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD |
| title_fullStr | A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD |
| title_full_unstemmed | A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD |
| title_short | A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for <i>In Vitro</i> Studies in AMD |
| title_sort | novel method of mouse rpe explant culture and effective introduction of transgenes using adenoviral transduction for i in vitro i studies in amd |
| topic | retina pigment epithelium explant culture RPE flatmount adenoviral transduction |
| url | https://www.mdpi.com/1422-0067/22/21/11979 |
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