Ex vivo Expansion of Bovine Corneal Endothelial Cells in Xeno-Free Medium Supplemented with Platelet Releasate

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.

Fetal bovine serum-free culture of endothelial progenitor cells-progress and challenges

“…Platelet derivatives were also used for culturing corneal EC (CEC; Chou, Burnouf, & Wang, ), known for their very limited proliferation capacity that makes ex vivo expansion a challenging task (Joyce, ). Platelet releasate (PR), prepared by calcium activation of platelet concentrates obtained from donor apheresis, was used with or without heat‐inactivation (HPR and PR, respectively; Chou et al, ). CEC were isolated from bovine eyes and cultured in DMEM:F12 medium supplemented with 5% FBS and additional components (the so‐called supplemented hormonal epithelial medium).…”

“…Increasing HPR concentration from 2.5% to 10% slightly improved cell growth and metabolic activity, and 10% HPR supplementation was used for further isolation of the primary CEC, which were shown to express EC phenotypic markers. Yet although this work demonstrated the feasibility of using human PRs as XF supplements for the isolation and ex vivo maintenance of CEC, it failed to demonstrate their potential for supporting long‐term expansion of these cells, which is particularly important for the development of cellular therapies for corneal blindness (Chou et al, ). Moreover, further studies are needed to test the effect of PRs on human CEC.…”

Animal models to assess the therapeutic efficacy of human serum and serum-converted platelet lysates for dry eye syndrome: Seeing is believing

“…In conclusion, therapeutic blood-derived materials such as serum, plasma, platelet lysates, as well as partially fractionated and virally-inactivated blood materials [8,29,30], that are candidates for the standardized treatment of DES, should be evaluated, once proven non toxic and efficient promoter of cell growth in vitro, in well-validated and established animal models. Such models can provide unique information to validate product effectiveness, product standardization, and optimize therapeutic strategy in humans affected by DES.…”

“…[5][6][7][8][9]. However, in the clinical field, it remains somewhat puzzling that blood-derived biomaterials are used, believed or claimed to be effective in a wide variety of applications although experimental evidence in animal model has not been always demonstrated, nor a definition of the optimal product composition for a given application provided [10].…”

International Forum on GMP‐grade human platelet lysate for cell propagation

“…We culture adipose‐tissue, bone marrow and Wharton jelly‐derived stem cells , as well as various cells such as corneal endothelial cells , human periodontal ligament cells, gingival fibroblasts, Chinese hamster ovary cells, periodontal ligament, gingival fibroblast cells , human embryonic kidney (HEK293A) fibroblast and rabbit corneal (SIRC) epithelial cell lines . For these HPL in 100% plasma, we typically use 5 to 10% (v/v) to supplement the basal media.…”