Abstract
Purpose :
Corneal stromal stem cells (CSSC) exhibit a potential to mediate wound healing. They are shown to prevent corneal scar formation in a mouse model of anterior stromal injury, through several mechanisms including the attenuation of lymphocytic infiltration to injured stromal tissue, and the production of regenerative cytokines and anti-scarring microRNAs to induce scarless healing and restore native-like stromal tissue. To produce donor CSSC for potential clinical use, we have developed Standard Operation Procedures (SOP) under Good Manufacturing Practice (GMP) to meet the regulatory compliance.
Methods :
We followed our standard protocol of donor corneal tissue processing and dissection to get anterior limbal stroma, which was then digested by GMP grade collagenase to obtain single cells with high cell viability. Using an xCELLigence system (Agilent) to detect electric impedance changes, primary cell attachment and cultivation was optimized with GMP alternatives showing non-toxicity and unaffected cell growth. The cryo-storage of GMP-expanded cells was tested using DMSO-free preservation media. Phenotypic characteristics of donor CSSC generated under GMP versus lab-based protocols were assessed by stem marker expression by qPCR and in vitro keratocyte differentiation by treating cells with basic fibroblast growth factor, transforming growth factor b3 and sodium ascorbate for 7 days followed by keratocyte marker expression analyses using immunofluorescence and qPCR.
Results :
A total of 22 donor corneas were used to obtain primary CSSC cultures in this GMP optimization study. We established the GMP compliant SOP using NB6 collagenase (Nordmark) for tissue digestion, recombinant human fibronectin (R&D Biosystems) for cell attachment, various GMP reagents in cell culture medium and DMSO-free StemCell Banker (Nippon Zenyaku) for cryopreservation. Thawed cells at passage 2 showed stem marker expression (ABCG2, NESTIN, BMI-1, PAX6 and CXCR4) at levels similar with cells generated under lab-based protocol. GMP cells also had upregulated expressions of keratocan, lumican, aquaporin 1, B3GnT7 and CHST6 after induced differentiation to keratocytes.
Conclusions :
We optimized GMP SOP produces clinical grade human CSSC and this development is a key to demonstrate the regulatory compliance and provide essential data for the Investigational New Drug (IND) application at The Food and drug Administration (FDA).
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.