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Biomolecules
Special Issues
Cell and Organ Cultures for Studying Retinal Diseases
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Cell and Organ Cultures for Studying Retinal Diseases

A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Molecular Medicine".

Deadline for manuscript submissions: closed (31 March 2022) | Viewed by 14879

Special Issue Editors

Prof. Dr. Stephanie C. Joachim

E-Mail Website
Guest Editor
Ophthalmology Department, Ruhr-Universitat Bochum, Bochum, Germany
Interests: glaucoma; age-related macular degeneration; retinal diseases; retinal organ culture models; autoimmunity; neuroprotection
Special Issues, Collections and Topics in MDPI journals
Dr. Sven Schnichels

E-Mail Website
Guest Editor
Centre for Ophthalmology, University Eye Hospital Tübingen, Tübingen, Germany
Interests: retinal organ culture models; glaucoma; retinal diseases; neuroprotection; ocular drug delivery; nanoparticles; ocular gene delivery; aptamers; AMD; IRD
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

For many retinal diseases, including age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy (DR), the exact pathogenesis is still unclear. Moreover, the currently available therapeutic options are often unsatisfactory.

To date, research regarding these diseases has mainly relied on animal models. However, animal models do not fully mimic human diseases. In addition, there is growing pressure from society to reduce animal research. This creates a need for alternative and improved disease models.

This Special Issue includes original research and review articles about recent developments regarding in vitro models for retinal diseases.

The topics of this Special Issue include but are not limited to:

  • The development of in vitro and ex vivo models (e.g., primary cell cultures, organotypic cultures, organoids, and whole eye cultures);
  • The analysis and validation of causative molecular pathways and pathomechanisms;
  • Treatment development and testing.

Prof. Dr. Stephanie C. Joachim
Dr. Sven Schnichels
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biomolecules is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Cell culture
  • Organ culture
  • Ex vivo
  • Retina culture
  • In vitro
  • Primary cell cultures
  • Organotypic cultures
  • Whole eye cultures
  • Organoids

Published Papers (6 papers)

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Research

22 pages, 3755 KiB  
Article
Impact of Primary RPE Cells in a Porcine Organotypic Co-Cultivation Model
by Natalie Wagner, Armin Safaei, José Hurst, Pia A. Vogt, H. Burkhard Dick, Stephanie C. Joachim and Sven Schnichels
Biomolecules 2022, 12(7), 990; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12070990 - 16 Jul 2022
Cited by 2 | Viewed by 2199
Abstract
The pathological events of age-related macular degeneration are characterized by degenerative processes involving the photoreceptor cells, retinal pigment epithelium (RPE), and the Bruch’s membrane as well as choroidal alterations. To mimic in vivo interactions between photoreceptor cells and RPE cells ex vivo, complex [...] Read more.
The pathological events of age-related macular degeneration are characterized by degenerative processes involving the photoreceptor cells, retinal pigment epithelium (RPE), and the Bruch’s membrane as well as choroidal alterations. To mimic in vivo interactions between photoreceptor cells and RPE cells ex vivo, complex models are required. Hence, the aim of this study was to establish a porcine organotypic co-cultivation model and enlighten the interactions of photoreceptor and RPE cells, with a special emphasis on potential neuroprotective effects. Porcine neuroretina explants were cultured with primary porcine RPE cells (ppRPE) or medium derived from these cells (=conditioned medium). Neuroretina explants cultured alone served as controls. After eight days, RT-qPCR and immunohistology were performed to analyze photoreceptors, synapses, macroglia, microglia, complement factors, and pro-inflammatory cytokines (e.g., IL1B, IL6, TNF) in the neuroretina samples. The presence of ppRPE cells preserved photoreceptors, whereas synaptical density was unaltered. Interestingly, on an immunohistological as well as on an mRNA level, microglia and complement factors were comparable in all groups. Increased IL6 levels were noted in ppRPE and conditioned medium samples, while TNF was only upregulated in the ppRPE group. IL1B was elevated in conditioned medium samples. In conclusion, a co-cultivation of ppRPE cells and neuroretina seem to have beneficial effects on the neuroretina, preserving photoreceptors and maintaining synaptic vesicles in vitro. This organotypic co-cultivation model can be used to investigate the complex interactions between the retina and RPE cells, gain further insight into neurodegenerative pathomechanisms occurring in retinal diseases, and evaluate potential therapeutics. Full article
(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)
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19 pages, 3077 KiB  
Article
Kir4.2 Potassium Channels in Retinal Pigment Epithelial Cells In Vitro: Contribution to Cell Viability and Proliferation, and Down-Regulation by Vascular Endothelial Growth Factor
by Marie-Christin Beer, Heidrun Kuhrt, Leon Kohen, Peter Wiedemann, Andreas Bringmann and Margrit Hollborn
Biomolecules 2022, 12(6), 848; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12060848 - 18 Jun 2022
Viewed by 1990
Abstract
Dedifferentiation and proliferation of retinal pigment epithelial (RPE) cells are characteristics of retinal diseases. Dedifferentiation is likely associated with changes of inwardly rectifying potassium (Kir) channels. The roles of Kir4.2 channels in viability, and proliferation of cultured RPE cells were investigated. Gene expression [...] Read more.
Dedifferentiation and proliferation of retinal pigment epithelial (RPE) cells are characteristics of retinal diseases. Dedifferentiation is likely associated with changes of inwardly rectifying potassium (Kir) channels. The roles of Kir4.2 channels in viability, and proliferation of cultured RPE cells were investigated. Gene expression levels were determined using qRT-PCR. RPE cells expressed Kir2.1, 2.2, 2.4, 3.2, 4.1, 4.2, 6.1, and 7.1 mRNA. Kir4.2 protein was verified by immunocytochemistry and Western blotting. Kir4.2 mRNA in cultured cells was upregulated by hypoxia (hypoxia mimetic CoCl2 or 0.2% O2) and extracellular hyperosmolarity (addition of high NaCl or sucrose). Kir4.2 mRNA was suppressed by vascular endothelial growth factor (VEGF), blood serum, and thrombin whereas platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-β1 (TGF-β1) increased it. Hyperosmotic Kir4.2 gene expression was mediated by TGF-β1 receptor signaling while hypoxic gene transcription was dependent on PDGF receptor signaling. VEGF receptor-2 blockade increased Kir4.2 mRNA level under control, hyperosmotic, and hypoxic conditions. SiRNA-mediated knockdown of Kir4.2 decreased the cell viability and proliferation under control and hyperosmotic conditions. Kir4.2 channels play functional roles in maintaining the viability and proliferation of RPE cells. Downregulation of Kir4.2 by VEGF, via activation of VEGF receptor-2 and induction of blood-retinal barrier breakdown, may contribute to decreased viability of RPE cells under pathological conditions. Full article
(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)
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16 pages, 3889 KiB  
Article
Impairment of the Retinal Endothelial Cell Barrier Induced by Long-Term Treatment with VEGF-A165 No Longer Depends on the Growth Factor’s Presence
by Heidrun L. Deissler, Matus Rehak and Armin Wolf
Biomolecules 2022, 12(5), 734; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12050734 - 23 May 2022
Cited by 2 | Viewed by 2006
Abstract
As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A165 depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes—determined as a [...] Read more.
As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A165 depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes—determined as a measure of permeability—was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A165 protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected. Nuclear levels of β-catenin were only elevated on day two. Extracellular levels of VEGF-A—measured by ELISA—were very low. Similar to the binding of the growth factor by brolucizumab, inhibition of VEGFR2 by tyrosine kinase inhibitors tivozanib or nintedanib led to complete, although transient, recovery of the low cell index when added early, though was inefficient when added three or six days later. Additional inhibition of other receptor tyrosine kinases by nintedanib was similarly unsuccessful, but additional blocking of c-kit by tivozanib led to sustained recovery of the low cell index, an effect observed only when the inhibitor was added early. From these data, we conclude that several days after the addition of VEGF-A165 to iBREC, barrier dysfunction is mainly sustained by increased paracellular flow and impaired adhesion. Even more important, these changes are most likely no longer VEGF-A-controlled. Full article
(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)
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18 pages, 3466 KiB  
Article
Impacts of Bacteriostatic and Bactericidal Antibiotics on the Mitochondria of the Age-Related Macular Degeneration Cybrid Cell Lines
by Nasim Salimiaghdam, Lata Singh, Mithalesh K. Singh, Marilyn Chwa, Shari R. Atilano, Zahra Mohtashami, Anthony B. Nesburn, Baruch D. Kuppermann, Stephanie Y. Lu and M. Cristina Kenney
Biomolecules 2022, 12(5), 675; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12050675 - 07 May 2022
Cited by 1 | Viewed by 2018
Abstract
We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. [...] Read more.
We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. Reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔψM), cellular metabolism and ratio of apoptotic cells were measured using H2DCFDA, JC1, MTT and flow cytometry assays, respectively. Expression of genes of antioxidant enzymes, and pro-inflammatory and pro-apoptotic pathways were evaluated by quantitative real-time PCR (qRT-PCR). Higher ROS levels were found in U haplogroup cybrids when treated with CPFX 60 µg/mL concentrations, lower ΔψM of all haplogroups by CPFX 120 µg/mL, diminished cellular metabolism in all cybrids with CPFX 120 µg/mL, and higher ratio of dead cells in K and J cybrids. CPFX 120 µg/mL induced overexpression of IL-33, CASP-3 and CASP-9 in all cybrids, upregulation of TGF-β1 and SOD2 in U and J cybrids, respectively, along with decreased expression of IL-6 in J cybrids. TETRA 120 µg/mL induced decreased ROS levels in U and J cybrids, increased cellular metabolism of treated U cybrids, higher ratio of dead cells in K and J cybrids and declined ΔψM via all TETRA concentrations in all haplogroups. TETRA 120 µg/mL caused upregulation of IL-6 and CASP-3 genes in all cybrids, higher CASP-7 gene expression in K and U cybrids and downregulation of the SOD3 gene in K and U cybrids. Clinically relevant dosages of ciprofloxacin and tetracycline have potential adverse impacts on AMD cybrids possessing K, J and U mtDNA haplogroups in vitro. Full article
(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)
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21 pages, 5089 KiB  
Article
Inherited Retinal Degeneration: PARP-Dependent Activation of Calpain Requires CNG Channel Activity
by Jie Yan, Alexander Günter, Soumyaparna Das, Regine Mühlfriedel, Stylianos Michalakis, Kangwei Jiao, Mathias W. Seeliger and François Paquet-Durand
Biomolecules 2022, 12(3), 455; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12030455 - 15 Mar 2022
Cited by 6 | Viewed by 2571
Abstract
Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). [...] Read more.
Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1−/− double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1−/− double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases. Full article
(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)
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19 pages, 1486 KiB  
Article
Complement Factor H Loss in RPE Cells Causes Retinal Degeneration in a Human RPE-Porcine Retinal Explant Co-Culture Model
by Angela Armento, Aparna Murali, Julia Marzi, Ana C Almansa-Garcia, Blanca Arango-Gonzalez, Ellen Kilger, Simon J Clark, Katja Schenke-Layland, Charmaine A Ramlogan-Steel, Jason C Steel and Marius Ueffing
Biomolecules 2021, 11(11), 1621; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11111621 - 03 Nov 2021
Cited by 6 | Viewed by 3098
Abstract
Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human [...] Read more.
Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H (CFH) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research. Full article
(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)
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