Thermoresponsive polymer surface coatings are a promising tool for cell culture applications. They allow for a mild way of cell detachment that preserves the activity of membrane proteins-a prerequisite for reliable cell analysis. To enlarge the application range of these coatings to cells with different adhesion properties, we synthesized various novel poly(ethylene glycol)-based thermoresponsive polymers and describe how (i) their chemical structure and (ii) their surface density affect their efficiency. In order to quantify the influence of both factors, the time for cell spreading and rounding efficiency were observed. As a result, efficiency of cell rounding, which is closely correlated to cell detachment, is less affected by both factors than the time needed for cell spreading. This time can effectively be adjusted by the molecular architecture which includes the length of the polymer backbone and the side chains. Based on this work, recommendations are given for future optimization of functionality of thermoresponsive polymer coatings for cell culture applications.