Evaluation of methods for cultivating limbal mesenchymal stromal cells

Bray, Laura J., Heazlewood, Celena F., Atkinson, Kerry, Hutmacher, Dietmar W., & Harkin, Damien G. (2012) Evaluation of methods for cultivating limbal mesenchymal stromal cells. Cytotherapy, 14(8), pp. 936-947.

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Background: Mesenchymal stromal cells (MSC) with similar properties to bone marrow derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We presently contribute to this novel area of research by evaluating methods for culturing human limbal MSC (L-MSC). Methods: Four basic strategies are compared: serum-supplemented medium (10% foetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor, and fibroblast growth factor 2, or one of two commercial serum-free media including Defined Keratinocyte Serum Free Medium (Invitrogen), and MesenCult-XF (Stem Cell Technologies). The phenotype of resulting cultures was examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141, CD271), immunocytochemistry (α-sma), differentiation assays (osteogenesis, adipogenesis, chrondrogenesis), and co-culture experiments with human limbal epithelial (HLE) cells. Results: While all techniques supported to varying degrees establishment of cultures, sustained growth and serial propagation was only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70-80% CD34-/CD45-/CD90+/CD73+/CD105+, approximately 25% α-sma+, and displayed multi-potency. Cultures established in MesenCult-XF were >95% CD34-/CD45-/CD90+/CD73+/CD105+, 40% CD141+, rarely expressed α-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3. Conclusions: We conclude that MesenCult-XF® is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.

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17 citations in Scopus
18 citations in Web of Science®
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ID Code: 53975
Item Type: Journal Article
Refereed: Yes
Keywords: cornea, epithelium, keratocytes, limbus, mesenchymal stromal cells
DOI: 10.3109/14653249.2012.684379
ISSN: 1465-3249
Subjects: Australian and New Zealand Standard Research Classification > MEDICAL AND HEALTH SCIENCES (110000)
Divisions: Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2012 Taylor & Francis
Copyright Statement: This is a preprint of an article submitted for consideration in the Cytotherapy © 2012 [copyright Taylor & Francis]; Cytotherapy is available online at: www.tandfonline.com
Deposited On: 04 Oct 2012 23:08
Last Modified: 23 Jun 2017 06:08

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