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Y.-H. Chen, I.-J. Wang, T.-H. Young; Culture Medium Affects Adhesive Behavior of Corneal Stromal Cells on Chitosan-Coated Surface and Tissue Culture Plate. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1874.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the difference of cell adhesive molecules between adhesive and spheroidic corneal stromal cells on chitosan and tissue-culture plate in two different medium.
Collagenase-digested bovine corneal stromal cells were seeded onto chitosan-coated plate (CM) and tissue culture plate (TCPS) with neural cell cultured medium (NCM) , which containing DMEM/F12, B27 supplement, EGF 10ng/ml, and bFGF 20ng/ml, or DMEM with 10% horse serum(HS). Cell viability of each group was determined by MTT assay. The mRNA of Keratin 3 (K3), keratocan, alpha-smooth muscle actin(alpha-SMA), and PAX6 were recognized by RT-PCR. To compare the cell adhesive force, RT-PCR was used to detect the cell adhesive molecules CD34, N-cadherin, cadherin 11, alpha5 integrin, and alpha v integrin.
Corneal stromal cell seeding on CM with DMEM/10% HS (C/H group) formed suspended cell-sphereoids. Moreover, cells attached to the TCPS with DMEM/10HS (T/H group) were dendritic shape. Cells in the NCM showed totally inverse adhesive property. Cells seeding on the CM with NCM (C/N group) surprisingly attached to the surface and showed morphologically smoother cell boundary than T/H group. Cells in NCM seeding on TCPS (T/N group) formed cell-spheroids and some of spheroids attached to the plates. To determine the cell activity, the results of MTT assay indicated that the attached cells ( T/H and C/N groups) showed higher mitochondrial activity than spheroids cells (C/H and T/N groups). RT-PCR results showed that cells under all four conditions presented keratocan-positive, alphaSMA-negative, and K3-negative phenotypes. In addition, PAX6 presented light positive for T/N and C/N groups and negative for T/H and C/H group. About the cell adhesive molecules, all groups expressed alph v integrin and cadhrin 11. In addition, only T/H and T/N groups showed CD34 positive and only C/N group expressed alpha 5 integrin negative.
Cell adhesive property and cell viability are different under different culture condition.
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