A photo- and thermoresponsive copolymer brush interface enabling label-free sequential separation of heterogeneous cell populations.

The label-free, sequential isolation of multiple cell types from heterogeneous populations remains challenging, as current sorting methods are limited in nondestructive collection, on-demand sequential control, and antibody-free processing. Herein, photo- and thermoresponsive copolymer brushes consisting of spiropyran methacrylate (SPMA) and di(ethylene glycol) methyl ether methacrylate (DEGMA) are reported as a platform to address these challenges. This platform supported cell adhesion at 37 °C comparable to tissue culture polystyrene. The polymer brushes exhibited distinct wettability changes upon combined photo and thermal stimulation compared with individual stimulus treatments. Fluorescent protein adsorption assays revealed a significant decrease in fluorescence intensity upon combined stimulation. Prolonged incubation at 22 °C further reduced the intensity. These interfacial changes correlated with differential cell detachment. PaTu 8988t cells were selectively detached from binary co-cultures within 10 min by mild rinsing. In ternary co-cultures, the P(SPMA-co-DEGMA) brushes enabled the sequential isolation of cancer cells, fibroblasts, and mesenchymal stem cells (MSCs). In this process, PaTu 8988t cells were first released by gentle rinsing after combined stimulation. Fibroblasts were subsequently detached by additional rinsing after a further 50 min incubation at 22 °C. MSCs remained adherent on the polymer brushes, resulting in their selective enrichment. The entire process was completed within 1 h without labeling. The detached cells maintained high viability and the ability to readhere. By correlating surface wettability changes and protein adsorption with differential cell detachment, this work provides insights into cell-surface interactions and offers a label-free strategy for sequential cell isolation.