Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

Abstract

Background Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities. Methods A total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring. Results By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring. Conclusions We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

1. Introduction

Corneal blindness is a leading cause of vision loss worldwide. Using the definition of blindness by World Health Organization, over 240 million people worldwide suffer from moderate-to-severe vision impairment due to corneal opacities, and about 10 million individuals have corneal blindness (https://www.who.int/news-room/fact-sheets/detail/blindness-and-visual-impairment). In some locations of Africa, nearly 90% of total blindness cases are related to corneal pathology [1]. Corneal opacification or scarring occurs after trauma, corneal ulcers, infections, surgeries or secondary to other corneal diseases or disorders, including congenital corneal dystrophies and degenerations (e.g., keratoconus and ectasia).

At present, safe and reliable treatments are limited. Topical corticosteroids and agents, such as mitomycin C, are used to treat or prevent corneal haze; however, there are variable efficacy and known adverse effects of these treatments. In addition, the safety, dosing and duration of these treatments are subjects of debate among clinicians. Corneal transplantation (penetrating and deep anterior lamellar keratoplasties) using donor corneas remains the most popular choice for replacement of scarred corneas in order to restore corneal functions and vision. About 185,000 corneal transplants are performed annually in 116 countries [2]. The limited global supply of transplantable donor corneas, immune response, risks of long-term graft failure, and the need for lifelong immunosuppression highlight the urgent need for alternative therapies.