Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Kalha, Solja; Kuony, Alison; Michon, Frédéric

doi:10.3791/58071-v

Cited by 4 publications

(2 citation statements)

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“…With the novel formula incorporating both the expression of stemness genes ABCG2 and nestin (ΔCT values after normalization with the housekeeping 18S) and the RInflam of three osteoclast genes, to calculate the Scarring Index (SI), we found that the healing potency of CSSCs can be predictable. The mouse model of mechanical anterior stromal injury created by Algerbrush burring has been deemed as the most effective way to mimic the traumatic injury in humans [ 35 ]. Inflicted by the rotating head of Algerbrush, the localized corneal wound with damaged Bowman’s membrane exhibits pronounced keratocyte apoptosis and activation of repair-type fibroblasts [ 36 , 37 ].…”

Section: Discussionmentioning

confidence: 99%

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Santra,

Geary,

Rubin

et al. 2024

Stem Cell Res Ther

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Background Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities. Methods A total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring. Results By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring. Conclusions We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

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Section: Discussionmentioning

confidence: 99%

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Santra,

Geary,

Rubin

et al. 2024

Stem Cell Res Ther

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“…Before the corneal punch procedure, the corneal epithelial wound was induced following a documented protocol. 13 Briefly, an ocular burr (Alloy Medical, San Mateo, CA, USA) was used to generate an abrasion on the central cornea after mice were anesthetized by an intraperitoneal injection of 50 mg/kg ketamine hydrochloride mixed with 5 mg/kg xylazine.…”

Section: Methodsmentioning

confidence: 99%

A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy

Liang,

Huang,

Yuan

et al. 2023

Invest. Ophthalmol. Vis. Sci.

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Purpose To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. Methods Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. Results Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. Conclusions The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.

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PCL scaffold combined with rat tail collagen type I to reduce keratocyte differentiation and prevent corneal stroma fibrosis after injury

Kong

Xie

et al. 2022

Experimental Eye Research

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The cornea is one of the major refractive eye components with signi cant functions, and its transparency is essential for clear vision. With regard to corneal injury, the corneal epithelium has a strong self-healing ability, while the corneal stroma is not capable of total self-repair. Therefore, preventing brosis and reducing keratocyte differentiation after injury have always been a challenge. The severe shortage of donor corneas for transplantation and transplant rejection prompted the development of corneal tissue engineering. In this study, we fabricated a poly(ε-caprolactone) (PCL) micro brous scaffold and infused the scaffold with rat tail collagen type I to obtain a 3D composite material. The PCL/collagen scaffold was designed to fabricate an optimal construct that simulates the stromal structure with properties that are most similar to the native cornea. The PCL scaffold has good mechanical properties, and infusion with rat tail collagen type I improved its biocompatibility. The results demonstrate that 3D composite material could reduce keratocyte differentiation, help achieve regular collagen distribution, and promote corneal repair.NanoSupplementarymaterialsandmethods.docx

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Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Cited by 4 publications

References 0 publications

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy

PCL scaffold combined with rat tail collagen type I to reduce keratocyte differentiation and prevent corneal stroma fibrosis after injury

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