In vitro evaluation of platelet extracellular vesicles (PEVs) for corneal endothelial regeneration

Abstract

Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 10¹¹ /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.

Keywords:

• Blood derived product
• corneal endothelial cells
• corneal endothelial dysfunction
• exosome
• platelet extracellular vesicles
• proteomics

Abbreviations

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Acknowledgements

The authors thank Taipei Blood Center for supplying platelet concentrates. We acknowledge the mass spectrometric technical services from the National Taiwan University (NTU) Consortia of Key Technologies and NTU Instrumentation Center. We thank Mr. Narpati Wesa Pikatan of the International PhD Program in Medicine, TMU, for his ind technical assistance to perform the Western blot analyse.

Disclosure statement

No potential conflict of interest was reported by the author(s)

Authors’ contributions

RW, TB, and TJW conceived the experimental design, discussed the results, and wrote the manuscript; RW acquired the data and performed the analysis. YWW, LD, and DYL provided scientific and technical assistance. All authors approved the final article.

Ethical approval and consent to participate

The Institutional Review Board of Taipei Medical University approved this study (TMU-JIRB no. 201802052)

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/09537104.2022.2105829

Additional information

Funding

The study was supported in part by a Taipei Medical University-Taipei Medical University Hospital research grant (108TMU-TMUH-03) from Taipei Medical University Hospital and the Ministry of Science and Technology of Taiwan (MOST 107-2314-B-038-032-MY2). The funding bodies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.