Abstract
Purpose We evaluated the suitability of 2% human platelet lysate (2%HPL) to replace 2% fetal bovine serum containing medium (2%FBS) for the xeno-free organ culture of human donor corneas.
Methods 32 human corneas unsuitable for transplantation from 16 human donors (age 69.3±15.7years) were collected 38.5±17.1 hours after death. They were first cultured in 2%FBS containing medium for 3 days (time point TP1), then evaluated by phase contrast microscopy (endothelial cell density (ECD) and cell morphology. Following an additional 25-days culture period (time point TP2) in either 2%FBS or 2%HPL medium the pairs were again compared by phase contrast microscopy (ECD and morphology), stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS).
Results ECD did not differ between the 2%HPL and 2%FBS group at TP1 (p=0.87). At TP2 the ECD was higher in the 2%HPL group (2179±288cells/mm2) compared to 2%FBS (2113±331cells/mm2; p=0.03), and endothelial cell loss was lower (ECL hPL=-0.7% vs. FBS=-3.8%; p=0.01). There were no significant differences in cell morphology, neither between TP1 and 2 nor between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial and 217 genes in stromal cells. 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (e.g. HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2) and the downregulation of pro-inflammatory/apoptotic genes (e.g. CXCL14, SIK1B, PLK5, PPP2R3B, SLURP1, FABP5, MAL, GATA3).
Conclusion 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and potentially beneficial alterations in gene expression.
