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Specific Assay Protocols for Porcine Single-Eye Retinal Pigment Epithelium Concerning Oxidative Stress and Inflammation.
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- Additional Information
- Source:
Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
- Publication Information:
Original Publication: Basel, Switzerland : MDPI, [2000-
- Subject Terms:
- Abstract:
The retinal pigment epithelium (RPE) is strongly involved in the pathogenesis of several retinal diseases, such as age-related macular degeneration (AMD). RPE models addressing specific pathological pathways are of high importance for understanding cellular pathomechanisms and pre-clinical screening of potential new therapeutics. The goal of this study is to establish standard operation protocols for single-eye porcine RPE preparation for AMD-relevant models of oxidative stress (RPE-Ox) and inflammation (RPE-Inf). Porcine primary RPE were prepared from one eye and seeded into one well of 12-well plates or, for polar differentiation, in transwell inserts. Different coatings (Poly-ᴅ-Lysine and laminin) and serum content of media (10%, 5%, and 1%) were tested to determine optimal culture parameters. For RPE-Ox, cells were treated with NaIO 3 , CoCl 2 , or erastin; cell viability (thiazolyl blue tetrazolium bromide, MTT), and gene expression (RT-qPCR) were determined. For RPE-Inf, cells were treated with lipopolysaccharide (LPS), polyinosinic/polycytidylic acid (Poly I:C), or tumor necrosis factor alpha (TNF-α); cell viability (MTT), cytokine secretion (ELISA), and gene expression (RT-qPCR) were determined. For transwell plates in RPE-Inf, cell viability (MTT), polar cytokine secretion (ELISA), gene expression (RT-qPCR), and transepithelial electrical resistance (TEER) for barrier assessment were conducted. For RPE-Ox, effective LD 50 could be achieved by using 24 h stimulation with 25 µm erastin, seven days after preparation in 5% serum cultures, without coating. For gene expression assessment, the use of Poly-ᴅ-Lysine is recommended. For RPE-Inf, three days of LPS stimulation (1 µg/mL) showed effective cytokine activation with 5% serum on uncoated 12-well plates. Transwell plates are not recommended for cytokine secretion assessment. It can be used for cell barrier assays in which LPS also showed effective cell barrier decrease and gene expression assays. Two specific best practice protocols for the use of porcine single-eye cultures in AMD research concerning oxidative stress and inflammation with optimized parameters were established and are provided.
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- Grant Information:
03LW0148 German Federal Ministry of Education and Research
- Contributed Indexing:
Keywords: 3R principle; age-related macular degeneration treatment; best practice protocol; inflammation; oxidative stress; primary cell culture; retinal pigment epithelium; single-eye cultures
- Accession Number:
0 (Cytokines)
0 (Lipopolysaccharides)
O84C90HH2L (Poly I-C)
0 (Tumor Necrosis Factor-alpha)
- Publication Date:
Date Created: 20250913 Date Completed: 20250916 Latest Revision: 20250916
- Publication Date:
20250916
- Accession Number:
PMC12428338
- Accession Number:
10.3390/ijms26178434
- Accession Number:
40943357
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