Isolation of microvascular endothelial cells from cadaveric corneal limbus

Gillies, Peter J., Bray, Laura J., Richardson, Neil A., Chirila, Traian V. and Harkin, Damien G. (2015) Isolation of microvascular endothelial cells from cadaveric corneal limbus. Experimental Eye Research, 131 20-28. doi:10.1016/j.exer.2014.12.008

Author Gillies, Peter J.
Bray, Laura J.
Richardson, Neil A.
Chirila, Traian V.
Harkin, Damien G.
Title Isolation of microvascular endothelial cells from cadaveric corneal limbus
Journal name Experimental Eye Research   Check publisher's open access policy
ISSN 1096-0007
Publication date 2015-02
Year available 2014
Sub-type Article (original research)
DOI 10.1016/j.exer.2014.12.008
Open Access Status
Volume 131
Start page 20
End page 28
Total pages 9
Place of publication London, United Kingdom
Publisher Academic Press
Collection year 2015
Language eng
Formatted 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.
Keyword CD
Cell culture
Corneal limbus
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Published online 10 December 2014

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2015 Collection
School of Medicine Publications
Australian Institute for Bioengineering and Nanotechnology Publications
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Citation counts: TR Web of Science Citation Count  Cited 1 times in Thomson Reuters Web of Science Article | Citations
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