Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5-Overexpressing Human Corneal Endothelial Cells

2018 ◽  
Vol 7 (18) ◽  
pp. 1800398 ◽  
Author(s):  
Kyoung Woo Kim ◽  
Soo Jin Lee ◽  
Soo Hyun Park ◽  
Jae Chan Kim
Keyword(s):  
Endothelial Cells ◽  
Corneal Endothelium ◽  
Ex Vivo ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells

scholarly journals Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells

2021 ◽  
Author(s):  
Mohit Parekh ◽  
Hefin Rhys ◽  
Tiago Ramos ◽  
Stefano Ferrari ◽  
Sajjad Ahmad
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Treatment Options ◽  
Proliferative Capacity ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells ◽  
The Media

Abstract Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs), are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line – HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 hours. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labelling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options.

scholarly journals Investigating the Effects of Coenzyme Q10 on Human Corneal Endothelial Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Mitchell Titley ◽  
Sajjad Ahmad ◽  
Mohit Parekh
Keyword(s):  
Endothelial Cells ◽  
Coenzyme Q10 ◽  
Ex Vivo ◽  
Annexin V ◽  
Positive Control ◽  
Dead Cell ◽  
Apoptotic Cells ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells ◽  
Viable Cells

Purpose. To investigate the effects of Coenzyme Q10 (CoQ10) treatment on immortalised human corneal endothelial cells (HCEC-12). Methods. HCEC-12 cells were cultured in different concentrations of CoQ10 (0.1%, 0.2%, 0.5%, and 1.0%) and analysed using live/dead staining assay to determine appropriate concentration for subsequent experiments. Cells were pretreated with CoQ10 before inducing apoptosis by ethanol (EtOH) treatment for 30 seconds which was followed by posttreatment with CoQ10. Viable, apoptotic, and dead cell proportions were analysed using Annexin V-FITC immunofluorescence staining. Mitochondrial intensity and respiratory functions were also investigated using MitoTracker staining and a Seahorse XFe24 analyser, respectively. Results were compared to a positive control for apoptosis. The experiments were carried out in triplicates. Graphpad prism software was used for statistical analysis where p < 0.05 was deemed statistically significant. Results. CoQ10 treatment at 0.5% and 1% showed 92% and 30% viable cells compared with 0.1% and 0.2% that showed 96% and 94% viable cells, respectively ( p = 0.0562 ). 0.1% and 0.2% concentrations were, thus, used for subsequent experiments. Annexin V-FITC apoptotic analysis showed 2% at 0.1% and 3% at 0.2% of apoptotic cells ( p = 0.0824 ). Mitochondrial respiratory function and mitochondrial intensity increased in apoptotic cells following 0.1% CoQ10 treatment. Conclusion. 0.1% CoQ10 was found optimal for reducing apoptosis and increasing metabolic activity on human corneal endothelial cell line. These results support the need for further ex vivo studies to investigate the safety profile of CoQ10 as an antiapoptotic agent for human corneal endothelium.

scholarly journals Proliferation Increasing Genetic Engineering in Human Corneal Endothelial Cells: A Literature Review

2021 ◽  
Vol 8 ◽  
Author(s):  
Wout Arras ◽  
Hendrik Vercammen ◽  
Sorcha Ní Dhubhghaill ◽  
Carina Koppen ◽  
Bert Van den Bogerd
Keyword(s):  
Endothelial Cells ◽  
Genetic Engineering ◽  
Corneal Endothelium ◽  
Treatment Strategies ◽  
Proliferative Capacity ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells ◽  
Intracameral Injection ◽  
Major Bottleneck ◽  
Donor Shortage

The corneal endothelium is the inner layer of the cornea. Despite comprising only a monolayer of cells, dysfunction of this layer renders millions of people visually impaired worldwide. Currently, corneal endothelial transplantation is the only viable means of restoring vision for these patients. However, because the supply of corneal endothelial grafts does not meet the demand, many patients remain on waiting lists, or are not treated at all. Possible alternative treatment strategies include intracameral injection of human corneal endothelial cells (HCEnCs), biomedical engineering of endothelial grafts and increasing the HCEnC density on grafts that would otherwise have been unsuitable for transplantation. Unfortunately, the limited proliferative capacity of HCEnCs proves to be a major bottleneck to make these alternatives beneficial. To tackle this constraint, proliferation enhancing genetic engineering is being investigated. This review presents the diverse array of genes that have been targeted by different genetic engineering strategies to increase the proliferative capacity of HCEnCs and their relevance for clinical and research applications. Together these proliferation-related genes form the basis to obtain a stable and safe supply of HCEnCs that can tackle the corneal endothelial donor shortage.

scholarly journals Receptor interacting protein 3 kinase, not 1 kinase, through MLKL-mediated necroptosis is involved in UVA-induced corneal endothelium cell death

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Zhen Yu ◽  
Nikolaos E. Efstathiou ◽  
Victor S. M. C. Correa ◽  
Xiaohong Chen ◽  
Kenji Ishihara ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Corneal Endothelium ◽  
Tissue Injury ◽  
Ex Vivo ◽  
Solar Spectrum ◽  
Corneal Endothelial Cells ◽  
Interacting Protein ◽  
Uva Irradiation ◽  
Culture Model

AbstractUltraviolet (UV) is one of the most energetic radiations in the solar spectrum that can result in various tissue injury disorders. Previous studies demonstrated that UVA, which represents 95% of incident photovoltaic radiation, induces corneal endothelial cells (CECs) death. Programmed cell death (PCD) has been implicated in numerous ophthalmologic diseases. Here, we investigated receptor-interacting protein 3 kinase (RIPK3), a key signaling molecule of PCD, in UVA-induced injury using a short-term corneal endothelium (CE) culture model. UVA irradiation activated RIPK3 and mediated necroptosis both in mouse CE and primary human CECs (pHCECs). UVA irradiation was associated with upregulation of key necroptotic molecules (DAI, TRIF, and MLKL) that lie downstream of RIPK3. Moreover, RIPK3 inhibition or silencing in primary corneal endothelial cells suppresses UVA-induced cell death, along with downregulation of MLKL in pHCECs. In addition, genetic inhibition or knockout of RIPK3 in mice (RIPK3K51A and RIPK3−/− mice) similarly attenuates cell death and the levels of necroptosis in ex vivo UVA irradiation experiments. In conclusion, these results identify RIPK3, not RIPK1, as a critical regulator of UVA-induced cell death in CE and indicate its potential as a future protective target.

Culturing Discarded Peripheral Human Corneal Endothelial Cells From the Tissues Deemed for Preloaded DMEK Transplants

Cornea ◽  
2019 ◽  
Vol 38 (9) ◽  
pp. 1175-1181 ◽  
Author(s):  
Mohit Parekh ◽  
Vito Romano ◽  
Alessandro Ruzza ◽  
Stephen B. Kaye ◽  
Diego Ponzin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells

scholarly journals Human Corneal Endothelial Cells Expanded In Vitro Are a Powerful Resource for Tissue Engineering

2017 ◽  
Vol 14 (2) ◽  
pp. 128-135 ◽  
Author(s):  
Yongsong Liu ◽  
Hong Sun ◽  
Min Hu ◽  
Min Zhu ◽  
Sean Tighe ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Endothelial Cells ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells
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