Abstract
Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory recommendations to limit the use of xenogenic materials.
Highlights
The corneal endothelium, an essential part of the cornea, is composed of a unique thin, fragile monolayer of hexagonal cells that dwell in the innermost layer of the cornea
The content of platelet growth factors (Table 2) was significantly higher (p,0.001) in the Platelet Releasate (PR) and HPR compared to PPP, apart from the hepatocyte growth factor (HGF)
The most important differences were more glucose, calcium, TIBC, UIBC, and ferritin, and less potassium, phosphate, iron and folate in platelet releasates compared to fetal bovine serum (FBS)
Summary
The corneal endothelium, an essential part of the cornea, is composed of a unique thin, fragile monolayer of hexagonal cells that dwell in the innermost layer of the cornea. Corneal endothelial cells (CECs) are embryologically derived from the neural crest. They cover the posterior surface of Descemet’s membrane and make contact with the aqueous humor [1]. CECs constitute a physiological and tight intercellular barrier that pumps fluids across the cornea, regulating hydration of the corneal stroma [2], and contributes to maintaining transparency and clarity for optimal visual functions [1,3,4,5,6]. The critical shortage of adequate tissue quality limits the availability of donor corneas [9], supporting the development of ex vivo CEC expansion to improve the supply for clinical-grade transplantation
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