Limbal Stem Cell Deficiency (LSCD) is a very serious and painful disease that often results in impaired vision. Cultivation of limbal stem cells for clinical application is usually performed on carriers such as amniotic membrane or surgical fibrin gel. Transplantation of these grafts is associated with the risk of local postoperative infection that can destroy the graft and devoid therapeutic benefit. For this reason, electrospun scaffolds are good alternatives, as proven to mimic the natural cells surroundings, while their fabrication technique is versatile with regard to polymer functionalization and scaffolds architecture. This study considers the development of poly(ε-caprolactone) (PCL) immune-compatible and biodegradable electrospun scaffolds, comprising cefuroxime (CF) or titanium dioxide (TiO₂) active components, that provide both bactericidal activity against eye infections and support of limbal stem cells growth in vitro. The PCL/CF scaffolds were prepared by blend electrospinning, while functionalization with the TiO₂ particles was performed by ultrasonic post-processing treatment. The fabricated scaffolds were evaluated in regard to their physical structure, wetting ability, static and dynamic mechanical behaviour, antimicrobial efficiency and drug release, through scanning electron microscopy, water contact angle measurement, tensile testing and dynamic mechanical analysis, antimicrobial tests and UV-Vis spectroscopy, respectively. Human limbal stem cells, isolated from surgical remains of human cadaveric cornea, were cultured on the PCL/CF and PCL/TiO₂ scaffolds and further identified through immunocytochemistry in terms of cell type thus were stained against p63 marker for limbal stem cells, a nuclear transcription factor and cytokeratin 3 (CK3), a corneal epithelial differentiation marker. The electrospun PCL/CF and PCL/TiO₂ successfully supported the adhesion, proliferation and differentiation of the cultivated limbal cells and provided the antimicrobial effect against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans.

Since only few reported studies propose anti-vascular endothelial growth factor (anti-VEGF) delivery through electrospun scaffolds, this study greatly contributes to the potential prevention of patient’s vision loss, as it explores electrospun polycaprolactone (PCL) coated with anti-VEGF for the blockage of abnormal cornea vascularization. In terms of physicochemical properties, the biological component increased the PCL scaffold fiber diameter (by ~24%) and pore area (by ~82%), while ut slightly reduced its total porosity as the anti-VEGF solution filled the voids of the microfibrous structure. The addition of the anti-VEGF increased the scaffold stiffness almost three-fold at both strains of 5 and 10%, as well as its biodegradation rate (~36% after 60 days) with a sustained release profile after Day 4 of phosphate buffered saline incubation. In terms of scaffold application function, the PCL/Anti-VEGF scaffold proved to be more favorable for the adhesion of cultured limbal stem cells (LSCs); this was confirmed by the SEM images, where the cells showed flat and elongated conformations. Further support of the LSC growth and proliferation was confirmed by the identified p63 and CK3 markers after cell staining. These results demonstrate the advantageous effect of the surface-adsorbed anti-VEGF to stop vision loss and help damaged corneal tissue repair.

Limbal stem cells (LSCs) are of paramount importance in corneal epithelial tissue repair. The cornea becomes opaque in case of limbal stem cell deficiency (LSCD), which may cause serious damage to the ocular visual function. There are many techniques to restore damaged epithelium, one of which is the transplantation of healthy cultured LSCs, usually onto a human amniotic membrane or onto bio-based engineered scaffolds in recent years. In this study, melt electrospun polylactic acid (PLA) was modified by silk fibroin or gelatin and further cultured with LSCs originating from three different donors. In terms of physicochemical properties, both modifications slightly increased PLA scaffold porosity (with a significantly larger pore area for the PLA/gelatin) and improved the scaffolds’ swelling percentage, as well as their biodegradation rate. In terms of the scaffold application function, the aim was to detect/visualize whether LSCs adhered to the scaffolds and to further determine cell viability (total number), as well as to observe p63 and CK3 expressions in the LSCs. LSCs were attached to the surface of microfibers, showing flattened conformations or 3D spheres in the formation of colonies or agglomerations, respectively. All scaffolds showed the ability to bind the cells onto the surface of individual microfibers (PLA and PLA/gelatin), or in between the microfibers (PLA/silk fibroin), with the latter showing the most intense red fluorescence of the stained cells. All scaffolds proved to be biocompatible, while the PLA/silk fibroin scaffolds showed the highest 98% viability of 2.9 × 10⁶ LSCs, with more than 98% of p63 and less than 20% of CK3 expressions in the LSCs, thus confirming the support of their growth, proliferation and corneal epithelial differentiation. The results show the potential of these bio-engineered scaffolds to be used as an alternative clinical approach.

Background Limbal stem cells(LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Through a clinically applicable protocol,the collected data on LSCs at passage 1 were summarized: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency were analyzed by trypan blue staining); expression of putative stem cell markers by flow cytometry, immunofluorescence and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n=873) was performed, and only a small fraction <2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.