title: Isolation of microvascular endothelial cells from cadaveric corneal limbus.
abstract: Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF(+) cells formed aligned cell-to-cell 'trains' when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32-80 years) or duration in eye bank corneal preservation medium prior to use (3-10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.
authors.affiliation: School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4001, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland, 4059, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Gillies
authors.affiliation: School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4001, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland, 4059, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia; Max Bergmann Center of Biomaterials, Leibniz-Institut für Polymerforschung e.V., Hohe Straße 6, 01069, Dresden, Germany.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Bray
authors.affiliation: School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4001, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland, 4059, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia.
authors.forename: Neil A
authors.initials: NA
authors.lastname: Richardson
authors.affiliation: Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia; Faculty of Health Sciences, University of Queensland, Herston, Queensland, 4006, Australia; Faculty of Science and Engineering, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4001, Australia; Australian Institute of Bioengineering & Nanotechnology, The University of Queensland, St Lucia, Queensland, 4072, Australia; Faculty of Science, The University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors.forename: Traian V
authors.initials: TV
authors.lastname: Chirila
authors.affiliation: School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4001, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland, 4059, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address: d.harkin@qut.edu.au.
authors.forename: Damien G
authors.initials: DG
authors.lastname: Harkin
mesh_terms: D002102:Cadaver; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D016850:Limbus Corneae / Q000098:blood supply* / Q000166:cytology; D055806:Microvessels / Q000166:cytology*
keywords: CD31/PECAM-1; Cell culture; Corneal limbus; Magnetic assisted cell sorting (MACS); Microvascular endothelial cells; von Willebrand factor (vWF)