(601f) Uncovering the Extracellular Matrix with Thermoresponsive Microgels

The extracellular matrix, or ECM, remains a hidden biological interface between a cell and a substrate. One method for examining proteins, such as those in the ECM, is flow cytometry (FC), which is a fast, high throughput method of quantification. Since FC is a solution technique, it is necessary to dissociate the cells from the surface. Traditional cell harvesting methods, such as enzymatic digestion and physical scraping, damage the ECM proteins as well as the cell morphology. Recently it has been shown that a thermoresponsive polymer, poly(N-isopropyl acrylamide) or pNIPAM, can be used to harvest a sheet of cells without damaging the integrity of the ECM. Above its lower critical solution temperature (LCST), this polymer is relatively hydrophobic, and mammalian cells grown on pNIPAM-grafted surfaces act in a similar fashion as those grown on typical tissue culture polystyrene (TCPS): they proliferate into a confluent sheet. Below the LCST (i.e., room temperature) the polymer physically changes, becoming more hydrophilic and swelling. Furthermore, when the temperature of the cell culture is dropped below the LCST of the polymer, the cells will detach as an intact cell sheet. These intact cell sheets, however, are not ideal for investigation in a flow cytometer, which requires individual cells for quantification. In this work, we developed a novel method for single cell detachment using pNIPAM microgels on the order of 20-200 micrometers. After fabrication, these gels were examined to ensure pNIPAM's characteristic thermoresponse was maintained, followed by cell culture on the gels. From these results, we conclude that these microgels have the capability of giving us access to this buried biological