Cell sheet technology is a novel approach to prepare and harvest monolayer cell sheets by using poly N-isopropylacrylamide (PNIPAm) or (PNIPAm)-modified surfaces as thermo-responsive cell culture substrates. As the temperature is lowered from 37°C to 20 °C, the cultured cells detach spontaneously as the surface substrate changes from hydrophobic to hydrophilic. However, evidence showed that the cell metabolic processes were suppressed below 32 °C, which may result in low cell viability of the recovered cell sheet and subsequent inefficient clinical therapies. In this study, we describe a cyclic peptide, which is used as a chemo responsive linker between cells and a PNIPAAm-modified surface. The linker is engineered to have high affinity for cells and for the PNIPAAm-modified surface. Release of the cells is achieved by a mild chemical process that modifies the cyclic peptide linker, reducing its affinity to the polymer surface. We will describe a process to discover synthetic polymer NPs with affinity and selectivity for a specific peptide sequence. A polymer NP is used as the bait to capture a peptide that is presented by a phage library. Based on our pervious work, a lightly crosslinked (2%) synthetic polymer hydrogel NP comprised of N-isopropyl acrylamide (NIPAm) monomers and hydrophobic and carboxylic acid groups, was found to bind with high affinity to the cyclic peptide, KPCISFWQFWFGFCSS (P14C). The interaction between the polymer NP and the cyclic peptide (P14C) is ~ three-fold stronger than with the corresponding linear peptide (P14L). Appending RGD onto the cyclic peptides’ N-terminal allows cell capture by the synthetic polymer NP to harvest cell sheets (see figure below). Glutathione (GSH), which is not harmful to cells, is used to cleave the cyclic peptide to linear peptide at 37°C to release the cell sheets. In preliminary results, NPs were coated on a 96 well plate. After addition of P14C-RGD, the cells attached to the wells in the plate. Control wells (blank and BSA coated) had only a few cells attached). The result establishes that P14C-RGD could be used as a linker between cell sheets and PNIPAAm-modified polymer surfaces. Synthetic polymers with high affinity for the RGD modified cyclic peptide will be applied to induce cell sheet formation and harvesting at 37°C.

Peptides with selective affinity to polymers for harvesting the cell sheet

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.