Abstract
Purpose :
Limbal stem cell deficiency (LSCD) is a blinding condition that is treated with limbal epithelial stem cell transplantation. Existing cultivation systems often contain xenogenic products and do not conform to good manufacturing practices (GMP). We report the development of cultivated autologous limbal epithelial cell (CALEC) constructs free of xenogenic products under GMP conditions.
Methods :
Cadaveric corneal biopsies (3x3mm2) were enzymatically digested and cultured on plastic until confluence (P0). Cells were detached and characterized by colony forming efficiency (CFE) assay and flow cytometry biomarker analysis. At the end of P0, 5x104 cells were seeded on denuded human amniotic membrane (hAM) (P1). At confluence, cell count and imaging was performed in situ with EVOS®. Gram stain, sterility, endotoxin, mycoplasma and viability (LDH assay) testing of the P1 supernatant were performed.
Results :
In process development, the average cellular yield at P0 was 3.8±2.1x105 cells, n=85. P0 cells were positive for epithelial cell markers (CD49F, CD49E, CD326, CD318, CD340), negative for hematopoietic (CD3, CD14, CD16, CD19, CD20, CD56), pan-leukocyte CD45, and endothelial CD31 markers. P1 culture duration was 8.8±2.5 days,n=68. The success rate of generating a construct was 97%, n=70. In hypothermic storage, LDH release correlated with the proportion of dead cells where LDH <100 U/ml and <10% dead cells were present at 24 hours, while LDH >250 U/ml correlated with >50% dead cells at 144 hours. In the GMP lab, 37 biopsies were cultivated to P0; 35 cultures were seeded on hAM. One biopsy had insufficient growth and one had contamination. The mean duration of P0 was 10±4 days and average cell count of 5.8±2.4x105 cells. CFE was 5.9±3.7%, n=15. The mean duration of P1 was 7±1 days and final cell count of 7.2±2.1x105 cells. After cells reached confluence on hAM, constructs met release criteria under GMP conditions and tested negative for endotoxin, mycoplasma, Gram stain and microbial culture.
Conclusions :
We have developed a standardized ex vivo cultivation system for autologous limbal epithelial cells. This system conforms to GMP standards and is free of serum, antibiotics, and xenogenic feeder cells, thus, providing a suitable construct for human transplantation. CALEC is being evaluated in a Phase I/II clinical trial sponsored by NEI/NIH (clinicaltrial.gov NCT02592330).
This is a 2020 ARVO Annual Meeting abstract.