Examining cell adhesion and gene expression on P(MEO2MA-co-OEGMA) and PNIPAM thermoresponsive biomaterials with integrated proteins.
Cara Abecunas, Lauren S. Anderson, PhD, Christopher R. Anderson, PhD
Department of Chemical and Biomolecular Engineering, Lafayette College, Easton, Pennsylvania
Thermoresponsive polymer (TRP) substrates as in vitro cell culture platforms have recently gained popularity amongst tissue engineers and biomaterialists due to their ability to release cells without biochemical enzymes and therefore preserve cell-cell and cell-matrix contacts. Cells grown on TRPs are released from their culture surface by a simple change in temperature below the lower critical solution temperature (LCST) of the polymer. The LCST of P(MEO2MA-co-OEGMA) or PMO substrates is tunable by varying the copolymer ratio while commercially available PNIPAM (UpCellTM, Thermo Scientific) has a single LCST of 32°C. In this work, we investigate cell growth on four PMO brushes of varying LCSTs (28, 31, 34 and 37°C) as well as PNIPAM in serum-free conditions. Various proteins were incorporated into the brushes including vitronectin, fibronectin, a combination of the two, and fetal bovine serum as the control group. Using Nikon NIS Elements Basic Research, the circularity and spreading of mouse fibroblast cells were quantified from phase contrast images. RT-PCR was used to determine the amplification of particular genes (FN1, VTN, ITGB3, and ITGAV). Results showed that by incorporating various proteins into the brushes, circularity decreased and the number of cells adhered to the surface increased, suggesting improved adhesion. FBS demonstrated significantly higher circularity values and fewer cells adhered to the TRPs. PCR results showed a down regulation in all of the genes except ITGAV, an alpha subunit used to form dimers with beta subunits (including ITGB3). The down regulation of ITGB3 suggests that ITGAV may be forming a dimer with another beta subunit and thus, further experimentation needs to be completed to verify these results. The down regulation of vitronectin (VTN) and fibronectin (FN1) suggest cells do not need to amplify these genes due to successful attachment to the incorporated proteins on TRPs. Results of this study will be used to optimize cell culture conditions for various cell types under serum-free conditions.
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