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P. Russell, J. Z. Gasiorowski, J. Last, C. J. Murphy; Substrate Compliance Influences Properties of Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2419.
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To determine if substrate compliance (relative stiffness) will impact human trabecular meshwork (HTM) cellular behaviors.
Primary HTM cells were cultured from donor corneal buttons. Cells were plated onto to either (1) compliant hydrogels made of polyacrylamide or (2) standard tissue culture polystyrene (TCPS) or glass. The intrinsic compliance of HTM cells was measured by AFM. HTM cells were treated with either dexamethasone or vehicle and RNA was extracted. Real-time PCR was used to quantify mRNA changes in fibronectin, myocilin, and matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) expression.
HTM cells grown on the hydrogels were rounder, less spread-out and intrinsically more compliant than cells grown on stiffer substrates. The cells on the hydrogels had only 37% of the fibronectin mRNA than the cells on the plastic surfaces. When dexamethasone was added to the culture to biochemically induce a glaucoma-like phenotype, the level of fibronectin expression by the cells on the more compliant surfaces increased to 1.2 times the value of the cells on plastic. This result is consistent with the increased expression of fibronectin in glaucomatous HTM. Myocilin levels were 5 fold higher in cells on the hydrogels and this value increased to 20 fold when dexamethasone was present in the culture media compared to cells on TCPS. MMP-2 expression levels were not changed with alterations in compliance, but the addition of dexamethasone decreased expression three fold in both cases. In contrast MMP-3 was not influenced by dexamethasone, but its expression was approximately 5 fold higher on the polyacrylamide surfaces compared to TCPS.
Growth of HTM cells on relatively compliant hydrogels significantly influences a number of characteristics including physical properties as well as gene expression for extracellular matrix production and degradation. By incorporating biophysical parameters that mimic the native in vivo environment we will create improved cell culture systems that allow us to study and characterize HTM cell behaviors that more clearly represent the cells growing in the meshwork itself.
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