Development of a new xeno-free human corneal endothelial cells culture system based on Platelet Rich in Growth Factors.

Abstract

Purpose : The purpose of the present study is to develop a culture system for human corneal endothelial cells (hCEC) replacing the use of xenogeneic components by a standardized kit in Ophthalmology to obtain human Platelet Rich in Growth Factors (PRGF) and to evaluate the adequacy of this culture system by the study of functionality markers, Transendothelial electrical resistance (TER) and gene expression.

Methods : 12 corneas discarded for transplantation were used for this study 6 per each group. Corneal endothelium was dissected following the Schwalbe line and was conditioned for 2-7 days at 37 °C in a culture plate with 4 mL of PRGF or SBF culture medium:
PRGF Medium: Optimem I, 10 v/v% PRGF, 200 mg/L CaCl₂ and 10 U/mL penicillin, 10 µg/mL streptomycin.
SBF Medium: Optimem I, 8 v/v% fetal bovine serum (FBS), 200 mg/L CaCl₂, 0.3 mM ascorbic acid 2-phosphate, 0.04% chondroitin sulfate, 20 ng/mL nerve growth factor, 5 ng/mL epidermal growth factor and 10 U/mL penicillin, 10 µg/mL streptomycin.
Conditioned endothelium was digested with TrypLE for 90 min at 37 °C. After that, the loosened cells were centrifuged at 0.4 g for 10 min, the supernatant was removed and the cells were seeded on a culture plate previously treated with FNC coating mix®.
Cellular growth was assessed by phase contrast microscopy until confluence. Both hCECs cultured with basal or conventional medium were used for TER evaluation. Confluent cultures were fixed using ice-cold methanol for 10 min. Then, immunocytochemistry of major corneal endothelial markers (Na⁺/K⁺ ATPase, ZO-1, conexin-43 and vimentin) were performed. Additionally confluent cultures of hCECs were loosened with accutase and used for gene expression analysis for the same expression markers.

Results : hCECs obtained from both PRGF and SBF supplemented cultures proliferated until confluence showing positive staining for conexin-43, vimentin, ZO-1 and Na⁺/K⁺ ATPase, responsible these last two for endothelial barrier and pump functions, respectively. In addition, similar gene expression levels and TER values were observed in PRGF and SBF hCECs cultures.

Conclusions : hCECs can be obtained using a PRGF culture medium avoiding the use of animal derived substances.

This is a 2021 ARVO Annual Meeting abstract.

 

immunofluorescences of the hCECs cultured with PRGF xeno-free or SBF medium