Pre-Clinical Evaluation of Efficacy and Safety of Human Limbus-Derived Stromal/Mesenchymal Stem Cells with and without Alginate Encapsulation for Future Clinical Applications

**Background:** Corneal scarring from infection, inflammation, or trauma is a major cause of blindness, often requiring corneal transplantation. However, donor cornea shortages and graft rejection limit this approach. Human limbus-derived stromal/mesenchymal stem cells (hLMSCs) have shown promise in preventing corneal haze in animal models. Alginate encapsulation may allow room-temperature transport, expanding access to cell therapy. This study aimed to evaluate the safety and efficacy of cGMP-manufactured hLMSCs, with and without alginate encapsulation, in pre-clinical models.

**Methods:** hLMSCs were isolated from 28 donor corneas and expanded in a GMP-compliant clean room. Cells were characterized by immunostaining (positive for p63-α, Pax6, ABCG2, CD73, CD90, CD105, VIM; negative for CD45) and FACS. Encapsulation used a sodium-alginate BeadReady™ kit; cells were transported at room temperature for 3–5 days. For efficacy, C57BL/6 mice (n=56) underwent central corneal debridement and were divided into scar group (treated 2 weeks post-wound), prophylaxis group (treated immediately), and untreated group. Subgroups received sham (vehicle), En- hLMSCs, or En+ hLMSCs (5×10⁴ cells in 2 μL fibrin glue). Clinical assessments included slit-lamp photography, OCT, and fluorescein staining over 28 days. For safety, New Zealand White rabbits (n=18; 3 groups of 6, 3M/3F) received sham (fibrin glue), En- hLMSCs (5×10⁵ cells), or En+ hLMSCs (50×10⁵ cells) after corneal scraping. Ophthalmic exams, IOP, blood and tear inflammatory markers (IL-6, TNF-α, IgE via ELISA), hematology, clinical chemistry, organ weights, and histopathology were assessed over 28 days.

**Key Results:** - **Efficacy:** In mice, corneal haze intensity decreased significantly in hLMSC-treated groups. In the scar group, haze dropped from 164±12 GSU (pre-treatment) to 121±6 GSU (En-) and 124±11 GSU (En+) at day 28 (p<0.0001). In the prophylaxis group, haze decreased from 151±14 to 138±19 GSU (En-) and 173±13 to 136±11 GSU (En+). Scar area reduced significantly in the scar group (p<0.001) but not in prophylaxis (p=0.0875). E:S reflectivity ratio normalized to baseline in hLMSC-treated eyes, while sham/untreated eyes showed increased stromal reflectivity (e.g., untreated: 0.96±0.01 to 0.65±0.02). - **Safety:** No mortality or clinical signs in rabbits. Ophthalmic exams showed only transient Grade 1 conjunctival inflammation at 3–6 h, resolving by 12 h. IOP remained normal and comparable across groups. Serum and tear IL-6 and TNF-α levels declined similarly in all groups. IgE levels were higher in En+ group at some time points but without clear trend. Hematology and clinical chemistry were largely normal; minor changes (e.g., phosphorus 7.35±1.11 mg/dL in G3 vs. 5.83±0.39 mg/dL in sham) were not associated with organ toxicity. Histopathology showed no dose-related adverse effects; sporadic findings (e.g., liver sinusoidal hemorrhages in 2/6 per group) were also present in controls. - **Cell stability:** hLMSCs maintained 88.33±2.37% viability at 6 h in pellet (2–8°C), doubling time <61 h, no chromosomal abnormalities, and endotoxin levels <0.12 EU/mL.

**Clinical Implications:** This pre-clinical study provides evidence that hLMSCs, with or without alginate encapsulation, are safe and effective in reducing corneal scarring in animal models. The lack of significant toxicity and inflammatory response supports progression to human clinical trials. Alginate encapsulation preserves cell efficacy and enables room-temperature transport, potentially reducing costs and improving access to cell-based therapy for corneal blindness, especially in resource-limited settings.