title: Evaluation of methods for cultivating limbal mesenchymal stromal cells.
abstract: OBJECTIVE
  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 have evaluated methods for culturing human limbal MSC (L-MSC).
  
  
  METHODS
  Four basic strategies were compared: serum-supplemented medium (10% fetal 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, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.
  
  
  RESULTS
  While all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were 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 multipotency. Cultures established in MesenCult-XF were > 95% CD34(-) CD45(-) CD90 (+) CD73 (+) CD105 (+) , 40% CD141 (+) , rarely expressed α-sma, and displayed multipotency. 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
  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.
authors.affiliation: Queensland Eye Institute, South Brisbane, Queensland, Australia. laura.bray@qut.edu.au
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Bray
authors.forename: Celena F
authors.initials: CF
authors.lastname: Heazlewood
authors.forename: Kerry
authors.initials: K
authors.lastname: Atkinson
authors.forename: Dietmar W
authors.initials: DW
authors.lastname: Hutmacher
authors.forename: Damien G
authors.initials: DG
authors.lastname: Harkin
mesh_terms: D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D003470:Culture Media; D005434:Flow Cytometry; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D003470:Culture Media