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Corinna Petsch, Ursula Schlotzer-Schrehardt, Markus Frey, Johannes Menzel-Severing, Friedrich Kruse, Bjoern Bachmann; Optimized culturing conditions for limbal epithelial cells cultivated on semi-synthetic collagen matrices. Invest. Ophthalmol. Vis. Sci. 2013;54(15):548.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize culturing conditions for limbal epithelial cells on variants of a semi-synthetic collagen matrices with different in vivo degradation characteristics.
Limbal epithelial stem cells were clonally enriched on 3T3 feeder cells and subcultivated on 3 variants (two crosslinked and one non-crosslinked variant) of a semi-synthetic type I collagen substrate (RESORBA, Germany). For clonal enrichment and subcultivation on the collagen matrices 3 different cell culture media were evaluated: MCDB151, DMEM/F12-1 (with bovine pituitary gland extract) and DMEM/F12-2 (without bovine pituitary gland extract). After fixation cell cultures were examined concerning cell adhesion, proliferation and cellular phenotype by light and electron microscopy as well as immunohistochemistry.
Immunohistochemistry as well as light and electron microscopy revealed no differences in adhesion, proliferation and cell sheet formation between cell cultures on either variant of the collagen matrix. When cultured with MCDB151 or DMEM/F12-1 monolayer formation of limbal epithelial cells was seen, while the use of DMEM/F12-2 resulted in a multilayered cell sheet. By immunohistochemistry, E-Cadherin (as a marker for adherens junctions) and K3/12 (as a differentiation marker) were localized in all cell layers. Integrinα6 (marker for hemidesomsomes) and p63 (a putative stem cell marker) were expressed in the basal cell layer. P63 was also apparent in upper layers of cells cultured on non-crosslinked collagen matrices. Electron microscopically hemidesmosomes were seen on cells of the basal cell layer of cultures on either collagen substrate when cultured with DMEM/F12-1 or DMEM/F12-2.
Crosslinked and non-crosslinked variants of a semi-synthetic collagen matrix are suitable for the cultivation of limbal epithelial cells. The use of DMEM/F12-2 with either collagen matrix resulted in a multilayered cell sheet of cultured limbal epithelial cells. The ability to serve as a growth substrate for limbal epithelial cells and its known biocompatibility with the cornea indicates that the used collagen matrices might be suitable for cultivation and transplantation of ex vivo expanded limbal epithelial cells.
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