Although there is a great deal of research focused on cell sheet engineering from polymers such as poly(N-isopropyl acrylamide) (pNIPAM), the biocompatibility of pNIPAM surfaces and the nature of cellular detachment from this polymer is still unclear. The most extensive study of the mechanism of detachment proposed a two-step process, with a first (passive) phase involving hydration of pNIPAM chains, and the second (active) phase involving cellular metabolism. However, a number of studies performed successful cell sheet detachment from pNIPAM-grafted surfaces at low temperatures which calls this hypothesis into question. Furthermore, although it has been demonstrated that low-temperature cell sheet detachment using pNIPAM-grafted surfaces is less destructive than other methods of detachment, it has not been investigated if cell sheet detachment removes a portion of pNIPAM from the surfaces as well. It is essential to know if any fragments of the polymer are removed along with the cells, as small polymer fragments could have cytotoxic effects on the cells. This is especially important if these cells are used for the generation of tissues used for transplantation. In this work, the mechanism of cell detachment from pNIPAM coated surfaces is investigated by testing how temperature and presence of an adenosine triphosephase inhibitor affect cellular detachment. Surface initiated atom transfer polymerization (ATRP) was utilized to synthesize thermoresponsive atrpNIPAM surfaces. pNIPAM surfaces were labeled to assess whether cell sheet detachment from pNIPAM is accompanied by the removal of pNIPAM from the substrate itself. Using a semipermeable superstrate, cell sheets were transferred to a secondary culture dish to assess whether cell detachment resulted in any pNIPAM removal. In addition, the function of the transplanted bovine aortic endothelial cells was assessed by determining whether they would proliferate and grow on a new secondary substrate.