Isolation of microvascular endothelial cells from cadaveric corneal limbus

Gillies, Peter J., Bray, Laura J., Richardson, Neil A., Chirila, Traian V., & Harkin, Damien G. (2015) Isolation of microvascular endothelial cells from cadaveric corneal limbus. Experimental Eye Research, 131, pp. 20-28.

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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 fetal 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 to 80 years) or duration in eye bank corneal preservation medium prior to use (3 to 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.

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ID Code: 79477
Item Type: Journal Article
Refereed: Yes
Keywords: Corneal limbus, Microvascular endothelial cells, Magnetic assisted cell sorting, CD31/PECAM-1, von Willebrand Factor
DOI: 10.1016/j.exer.2014.12.008
ISSN: 0014-4835
Subjects: Australian and New Zealand Standard Research Classification > TECHNOLOGY (100000) > MEDICAL BIOTECHNOLOGY (100400) > Regenerative Medicine (incl. Stem Cells and Tissue Engineering) (100404)
Australian and New Zealand Standard Research Classification > MEDICAL AND HEALTH SCIENCES (110000) > OPTOMETRY AND OPHTHALMOLOGY (111300) > Ophthalmology (111301)
Divisions: Current > Schools > School of Biomedical Sciences
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2014 Elsevier Ltd.
Copyright Statement: NOTICE: this is the author’s version of a work that was accepted for publication in Experimental Eye Research. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Experimental Eye Research, [VOL 131, (2015)] DOI: 10.1016/j.exer.2014.12.008
Deposited On: 16 Dec 2014 00:53
Last Modified: 18 Mar 2016 03:12

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