Encapsulation of human limbus-derived stromal/mesenchymal stem cells for biological preservation and transportation in extreme Indian conditions for clinical use

Abstract

Human limbus-derived stromal/mesenchymal stem cells (hLMSC) can be one of the alternatives for the treatment of corneal scars. However, reliable methods of storing and transporting hLMSC remains a serious translational bottleneck. This study aimed to address these limitations by encapsulating hLMSC in alginate beads. Encapsulated hLMSC were kept in transit in a temperature-conditioned container at room temperature (RT) or stored at 4 °C for 3–5 days, which is the likely duration for transporting cells from bench-to-bedside. Non-encapsulated cells were used as controls. Post-storage, hLMSC were released from encapsulation, and viability-assessed cells were plated. After 48 and 96-hours in culture the survival, gene-expression and phenotypic characteristics of hLMSC were assessed. During transit, the container maintained an average temperature of 18.6 ± 1.8 °C, while the average ambient temperature was 31.4 ± 1.2 °C (p = 0.001). Encapsulated hLMSC under transit at RT were recovered with a higher viability (82.5 ± 0.9% and 76.9 ± 1.9%) after 3 (p = 0.0008) and 5-day storage (p = 0.0104) respectively as compared to 4 °C (65.2 ± 1.2% and 64.5 ± 0.8% respectively). Cells at RT also showed a trend towards greater survival-rates when cultured (74.3 ± 2.9% and 67.7 ± 9.8%) than cells stored at 4 °C (54.8 ± 9.04% and 52.4 ± 8.1%) after 3 and 5-days storage (p > 0.2). Non-encapsulated cells had negligible viability at RT and 4 °C. Encapsulated hLMSC (RT and 4 °C) maintained their characteristic phenotype (ABCG2, Pax6, CD90, p63-α, CD45, CD73, CD105, Vimentin and Collagen III). The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in transit at RT, therefore, potentially expanding the scope of cell-based therapy for corneal blindness.

Human Cadaveric Donor Cornea Derived Extra Cellular Matrix Microparticles for Minimally Invasive Healing/Regeneration of Corneal Wounds

“…Human corneal stem cells (hCSCs) were isolated and expanded as per our previously published protocol [ 20 ]. Briefly, hCSCs were isolated from collagenase digested limbal stromal tissue of human corneal rims from which central tissue had been removed.…”

Hypothermically Stored Adipose-Derived Mesenchymal Stromal Cell Alginate Bandages Facilitate Use of Paracrine Molecules for Corneal Wound Healing

“…To restore corneal transparency and integrity, Ad-MSCs therapy has been investigated [ 35 , 36 , 37 , 38 , 39 ]. Using clinical and animal studies, Ad-MSCs therapy has recently proven to be a promising method used in corneal reconstruction with multiple treatment approaches [ 40 , 41 , 42 , 43 ]. Moreover, it has been found that Ad-MSCs are able to provide paracrine factors in response to the wound but also able to avoid host immune rejection and transdifferentiate into multilineages [ 10 , 17 , 44 ].…”

“…To restore corneal transparency and integrity, Ad-MSCs therapy has been investigated [35][36][37][38][39]. Using clinical and animal studies, Ad-MSCs therapy has recently proven to be a promising method used in corneal reconstruction with multiple treatment approaches [40][41][42][43].…”

Safety and toxicity of human limbus-derived stromal/mesenchymal stem cells with and without alginate encapsulation for clinical application

“…Cells were cultured on 18mm diameter coverslips in 12-well culture plates at a density of 20,000 cells/cm 2 at 37°C with 5% CO 2 in a humidi ed incubator until con uence. These cells were assessed for the expression of characteristic biomarkers of the mesenchymal stem cell (MSC) phenotype, as described previously (25). The antibody panel was composed of (a) ABCG2, Pax6, p63-α and Col-III as positive markers of the human limbal stem cell phenotype; (b) VIM, CD73, CD90, and CD105 as positive markers of the mesenchymal phenotype, and CD45 as a negative marker for mesenchymal origin.…”

“…The hLMSCs could potentially lower the need for corneal transplantation, therefore reducing the need for donor corneas. It has also been shown that encapsulation of hLMSCs in sodium alginate, retains their phenotype and maintains the viability, while being stored or transported at varied temperatures and for prolonged durations (3-5 days) (25). This easy-to-use technology, which obviates the need for an expensive cold chain, could increase the accessibility of hLMSC-therapy, particularly in remote geographical locations, without requiring the patient to travel hundreds of miles, particularly in developing countries.…”