Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities (original) (raw)
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Investigative Ophthalmology & Visual Science, 2006
To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. METHODS. Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-m depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. RESULTS. Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and ␣-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. CONCLUSIONS. The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy. (Invest Ophthalmol Vis Sci. 2006;47:552-557)
Irreversible optical clearing of rabbit dermis for autogenic corneal stroma transplantation
Biomaterials, 2011
Tissue engineering and transplantation of autogenic grafts have been widely investigated for solving problems on current allograft treatments (i.g., donor shortage and rejection). However, it is difficult to obtain an autogenic corneal stromal replacement that is composed of transparent, tough, and thick collagen constructs by current cell culture-based tissue engineering. Aim of this study is to develop transparent dermis for an autogenic corneal stroma transplantation. This study examined dehydration at 4e8 C and carbodiimide cross-linking on cloudy rabbit dermis (approx. 1.8%e3.8% light transmittance at 550 nm) for dermis optical clearing. Transparency of dehydrated rabbit dermis was founded to be approx. 37.9%e41.4% at 550 nm. Additional cross-linking treatment on dehydrated dermis prevented from swelling and clouding in saline, and improved its transparency to be 56.9% at 550 nm. Rabbit corneal epithelium was found to regenerate on optically cleared dermis in vitro. Furthermore, no abnormal biological response (i.e., inflammation, vascularization, and the barrier defect of epithelia) or no optical functional change on optically cleared dermis was observed during its 4-week autogenic transplantation into rabbit corneal stromal pocket.
An Attempt to Construct the Stroma of Cornea Using Primary Cultured Corneal Cells
Journal of Nanoscience and Nanotechnology, 2007
The number of patients currently awaiting corneal transplantation has resulted in the need to develop an artificial corneal replacement. In this study, we aimed to construct the corneal stroma using non-transformed corneal cells and a perfusion cell culture method. Corneal cells isolated from chicken embryos or rabbit and were embedded in the alkaline solubilized collagen gels crosslinked by TSG (Pentaerythritol polyethyleneglycol ether tetrasuccinimidyl glutarate). During culture, the majority of cells migrated from inside of the gel. The chicken and rabbit cells changed their morphology and stratified structures were constructed within the gels. These microstructures were similar to the natural corneal tissue. TEM analysis was performed to confirm the nano-microstructure of the constructs. Contrary to expectation, the cornea-like nanostructure of collagen fibrils was not observed within the gels. Further study including for example, such as the addition of dynamic stress or co-culture with endothelial cells, are therefore required in order to produce artificial constructs with the same superstructure as natural corneal tissue.
Stem cell therapy restores transparency to defective murine corneas
Stem cells (Dayton, Ohio), 2009
Corneal scarring from trauma and inflammation disrupts vision for millions worldwide, but corneal transplantation, the primary therapy for corneal blindness, is unavailable to many affected individuals. In this study, stem cells isolated from adult human corneal stroma were examined for the ability to correct stromal opacity in a murine model by direct injection of cells into the corneal stroma. In wild-type mice, injected human stem cells remained viable for months without fusing with host cells or eliciting an immune T-cell response. Human corneal-specific extracellular matrix, including the proteoglycans lumican and keratocan, accumulated in the treated corneas. Lumican-null mice have corneal opacity similar to that of scar tissue as a result of disruption of stromal collagen organization. After injection with human stromal stem cells, stromal thickness and collagen fibril defects in these mice were restored to that of normal mice. Corneal transparency in the treated mice was ind...
Ophthalmic Research
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 μm 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-...
Corneal stromal stem cells restore transparency after N 2 injury in mice
STEM CELLS Translational Medicine, 2020
Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N2) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early infla...
Nature Biomedical Engineering, 2019
Dysfunction of the corneal endothelium reduces the transparency of the cornea and can cause blindness. Because corneal endothelial cells have an extremely limited proliferative ability in vivo, treatment for corneal endothelial dysfunction involves the transplantation of donor corneal tissue. Corneal endothelium can also be restored via intraocular injection of endothelial cells in suspension after their expansion in vitro. Yet, because quality assessment during the expansion of the cells is a destructive process, a substantial number of the cultured cells are lost. Here, we show that the 'spring constant' of the effective interaction potential between endothelial cells in a confluent monolayer serves as a biomarker of the quality of corneal endothelial cells in vitro and of the long-term prognosis of corneal restoration in patients treated with culture-expanded endothelial cells or with transplanted corneas. The biomarker can be measured from phase contrast imaging in vitro and from specular microscopy in vivo, and may enable a shift from passive monitoring to pre-emptive intervention in patients with severe corneal disorders.