Abstract
Purpose Comprehensible concerns have been raised regarding the safety of FBS-based culture media. In this talk we discuss the benefits of using human platelet lysate (HPL) for the xeno-free culture of human donor corneas, isolated corneal stromal keratocytes (CSK) and stromal fibroblasts (SF).
Methods 32 human corneas unsuitable for transplantation from 16 human donors were cultured for 25-days in either 2%FBS or 2%HPL medium and compared by phase contrast microscopy (ECD and morphology), and next generation sequencing (NGS). Effects of 0.5%FBS, 5%FBS, 0.5%HPL, 2%HPL and 10%hPL on cultured human CSK and SF were evaluated.
Results Differential cornea culture showed lower endothelial cell loss in the 2%HPL vs. 2%FBS group (ECL hPL=-0.7% vs. FBS=-3.8%; p=0.01). 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (e.g. HMOX1, SERPINE1, ANGPTL4, LEFTY2) and the downregulation of pro-inflammatory/apoptotic genes (e.g. CXCL14, SIK1B, PLK5, PPP2R3B). CSK/SF cell viability remained high in all groups (98-100%). Cell numbers and proliferation rates increased (p=0.024-0.001), CSK marker expression decreased with higher fractions of HPL and FBS (p<0.001). SMA1 increased with higher amounts of FBS (p=0.003) but decreased with incremental HPL substitution in both cell types (p=0.014). HPL contained more TGF-β1 (100%hPL 1.861±0.231ng/ml vs. 100%FBS 0.015±0.010ng/ml, p<0.001). bFGF and HGF were only detectable in 100% hPL (bFGF 0.067±0.017ng/ml, HGF 1.074±0.050ng/ml).
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. CSK and SF can be cultured with xeno-free hPL. To maintain CSK characteristics substitution must remain minimal (0.5% hPL/FBS). hPL contains the antifibrotic HGF und bFGF, suppressing myofibroblast conversion.
