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Preprints

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

Damala M, Pakalapati N, Singh V, Basu S.
Preprint from
Research Square
11 August 2021
PPR
PPR381803
Abstract

Background:

The purpose of this study was to assess the ocular and systemic toxicity of topically applied human limbus-derived stromal/mesenchymal stem cells (hLMSCs) with and without alginate encapsulation as per Indian regulatory guidelines for stem cell therapy. Methods The hLMSCs were obtained from cadaveric corneoscleral rims and expanded in a current good manufacturing practice compliant laboratory. The hLMSCs were checked for viability, chromosomal stability, growth kinetics, contamination, and endotoxin levels. Cells with ( En + hLMSCs) or without ( En − hLMSCs) alginate encapsulation were used for the animal experiments. The study involved 3 groups of 6 New Zealand white rabbits each, which underwent corneal wounding followed by treatment with sham (G1), En − hLMSCs (G2), and En + hLMSCs cells (G3). Ophthalmic assessment including intraocular pressure (IOP), blood investigations and inflammatory marker (IL-6, TNF-α, IgE) expression in serum and tears were assessed on days 1, 7, 14, 21, and day 28. At the end of 28 days, the animals were sacrificed, and the organs were subjected to histopathological examination. Results The hLMSCs had 88.33 ± 2.37% viability at the end of 6 hours and 78.21 ± 1.47% at the end of 24 hours. The cells showed positive expression for the stem-cell biomarkers (p63α, Pax6, and ABCG2), extracellular matrix marker (Col-III) and mesenchymal biomarkers (VIM, CD73, CD90 and CD105). No contamination by the Mycoplasma species was found in either of the En- / En +  hLMSCs and the levels of bacterial endotoxins in the En- hLMSCs and En +  hLMSCs cell suspension was found be within the permissible levels (≤ 0.12 EU/mL). Ophthalmic examination showed no significant difference in IOP, corneal clarity and conjunctival congestion between the three groups at every time point. Haematological parameters were comparable between the three groups. The inflammatory markers in tear and serum (TNF-α and IL-6) were not significantly elevated in the groups receiving En + /En − hLMSCs. Histological examination did not show any abnormality in the ocular or corneal tissue, and the viscera. Conclusions The results of the study show that hLMSCs do not cause any local or systemic toxicity in recipients, implying that these cells are safe for clinical use and their efficacy can be assessed in human clinical trials.