Effects of human platelet lysate on the growth of cultured human corneal endothelial cells

Constantinos Petsoglou; Li Wen; Monira Hoque; Meidong Zhu; Monika Valtink; Gerard Sutton; Jingjing You

doi:10.1016/j.exer.2021.108613

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells

Exp Eye Res. 2021 Jul:208:108613. doi: 10.1016/j.exer.2021.108613. Epub 2021 May 10.

Authors

Constantinos Petsoglou¹, Li Wen², Monira Hoque², Meidong Zhu³, Monika Valtink⁴, Gerard Sutton¹, Jingjing You⁵

Affiliations

¹ Discipline of Ophthalmology, Sydney Medical School, The University of Sydney, Camperdown, NSW, 2006, Australia; New South Wales Tissue Bank, New South Wales Organ and Tissue Donation Service, Sydney, NSW, 2000, Australia; Save Sight Institute, The University of Sydney, Sydney, NSW, 2000, Australia; Corneal Unit, Sydney Eye Hospital, Sydney, NSW, 2000, Australia.
² New South Wales Tissue Bank, New South Wales Organ and Tissue Donation Service, Sydney, NSW, 2000, Australia.
³ New South Wales Tissue Bank, New South Wales Organ and Tissue Donation Service, Sydney, NSW, 2000, Australia; Save Sight Institute, The University of Sydney, Sydney, NSW, 2000, Australia. Electronic address: meidong.zhu@health.nsw.gov.au.
⁴ Institute of Anatomy, Faculty of Medicine, TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
⁵ Save Sight Institute, The University of Sydney, Sydney, NSW, 2000, Australia.

PMID: 33984343
DOI: 10.1016/j.exer.2021.108613

Abstract

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

Keywords: Fibrinogen; Human corneal endothelial cells; Human platelet lysate; IncuCyte ZOOM.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adult
Aged
Aged, 80 and over
Blood Platelets*
Cell Differentiation
Cell Proliferation
Cells, Cultured
Endothelium, Corneal / cytology
Endothelium, Corneal / growth & development*
Female
Humans
Male
Middle Aged