RESUMEN
Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.
Asunto(s)
Diferenciación Celular , Queratocitos de la Córnea , Opacidad de la Córnea , Animales , Masculino , Opacidad de la Córnea/patología , Ratas , Queratocitos de la Córnea/metabolismo , Humanos , Modelos Animales de Enfermedad , Sustancia Propia/metabolismo , Sustancia Propia/ultraestructura , Sustancia Propia/efectos de los fármacos , Agudeza Visual , Recuperación de la Función , Córnea/patología , Córnea/metabolismo , Ratas Sprague-DawleyRESUMEN
The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.
Asunto(s)
Separación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Queratocitos de la Córnea/citología , Sustancia Propia/citología , Ingeniería de Tejidos , Proliferación Celular , Forma de la Célula , Células Cultivadas , Queratocitos de la Córnea/metabolismo , Criopreservación , Regulación de la Expresión Génica , HumanosRESUMEN
With the expected rise in patients undergoing refractive lenticule extraction worldwide, the number of discarded corneal stromal lenticules will increase. Therefore, establishing a lenticule bank to collect, catalog, process, cryopreserve, and distribute the lenticules (for future therapeutic needs) could be advantageous. In this study, we validated the safety of lenticule banking that involved the collection of human lenticules from our eye clinic, transportation of the lenticules to a Singapore Ministry of Health-licensed lenticule bank, processing, and cryopreservation of the lenticules, which, after 3 months or, a longer term, 12 months, were retrieved and transported to our laboratory for implantation in rabbit corneas. The lenticule collection was approved by the SingHealth Centralised Institutional Review Board (CIRB). Both short-term and long-term cryopreserved lenticules, although not as transparent as fresh lenticules due to an altered collagen fibrillar packing, did not show any sign of rejection and cytotoxicity, and did not induce haze or neovascularization for 16 weeks even when antibiotic and steroidal administration were withdrawn after 8 weeks. The lenticular transparency progressively improved and was mostly clear after 4 weeks, the same period when we observed the stabilization of corneal hydration. We showed that the equalization of the collagen fibrillar packing of the lenticules with that of the host corneal stroma contributed to the lenticular haze clearance. Most importantly, no active wound healing and inflammatory reactions were seen after 16 weeks. Our study suggests that long-term lenticule banking is a feasible approach for the storage of stromal lenticules after refractive surgery. Impact statement Since 2011, close to 3 million refractive lenticule extraction procedures have been performed. The majority of the extracted lenticules are discarded. The lenticules could have been cryopreserved and retrieved at a later date for therapeutic or refractive applications. Therefore, establishing a lenticule bank to collect, catalog, process, cryopreserve, and distribute the lenticules could be advantageous. In this study, we simulated a lenticule banking service in a validated health authority-licensed facility and showed that long-term cryopreservation of the lenticules in the facility was safe and feasible in vivo.