Corneal endothelial cell therapy: feasibility of cell culture from corneas stored in organ culture

Hé, Zhiguo; Okumura, Naoki; Sato, Masakazu; Komori, Yuya; Nakahara, Makiko; Gain, Philippe; Koizumi, Noriko; Thuret, Gilles

doi:10.1007/s10561-021-09918-8

Cited by 9 publications

(8 citation statements)

References 35 publications

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“…In particular, once CHIR was added to HCEnCs that had already underwent EnMT, cell morphology, as measured by cell circularity 22 , was maintained through the passages up to passage 8, while α-SMA was significantly downregulated both at RNA and protein level and CD44, another marker of EnMT 60 , was significantly downregulated at RNA level. CEnCs markers such as Na + /K + ATPase, N-cadherin and ZO-1 61 , were also preserved upon CHIR treatment. The same result was obtained by adding CHIR in HCEnCs that still maintained a normal hexagonal phenotype.…”

Section: Discussionmentioning

confidence: 95%

GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells

Maurizi

Merra

Macaluso

et al. 2022

Preprint

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Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of alpha-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed CHIR99021 induced downregulation of EnMT markers (alpha-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the beta-catenin and TGFbeta; pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT and brings a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.

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Section: Discussionmentioning

confidence: 95%

GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells

Maurizi

Merra

Macaluso

et al. 2022

Preprint

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“…Other studies have also been able to establish viable hCEC cultures from elderly donors, both in cornea preserved at low temperature and in organotypic culture conditions [ 10 , 37 ]. The latter can maintain corneal tissue in optimal conditions for up to 3 or 4 weeks, offering considerable logistical advantages with respect to cold preservation methods.…”

Section: Discussionmentioning

confidence: 99%

Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy

Aloy-Reverté,

Bandeira,

Otero

et al. 2023

Ophthalmic Res

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Introduction: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. Methods: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 μ<sc>m</sc> Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. Results: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. Conclusions: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

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“…Human corneas were obtained from an 86-year-old donor with a short time between death and procurement (9 h). Corneal endothelial cells were cultured according to previously published protocol [ 27 ]. Briefly, the endothelium was stained with 0.4% trypan blue (Eurobio Scientific, Paris, France) and mechanically peeled off from the cornea.…”

Section: Methodsmentioning

confidence: 99%

Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering

Ben Moussa,

Parveau,

Aouimeur

et al. 2024

Bioengineering

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The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts.

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Corneal endothelial cell therapy: feasibility of cell culture from corneas stored in organ culture

Cited by 9 publications

References 35 publications

GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells

GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells

Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy

Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering

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