Application of PNIPAM-Based Microstructured Surfaces for Label-Free Cell Sorting

Cell sorting enables the separation of specific cell types from heterogeneous populations and is widely used in biomedical research, drug development, and regenerative medicine. Existing methods fall into two categories: label-based techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), which rely on antibodies conjugated to fluorescent dyes or magnetic beads; and label-free techniques, which exploit intrinsic physical properties such as size, deformability, or electrical charge. Although label-based methods offer high specificity, antibody labeling carries significant drawbacks, including elevated cost, risk of contamination, and potential interference with cell signaling, proliferation, and post-transplant immune responses.

We propose an antibody-free cell sorting platform based on differences of cell adhesion, exploiting variation in the type and density of adhesion molecules expressed on the surface of distinct cell types. Our approach utilizes microstructured (MS) surfaces composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) blended with polystyrene (PS) or poly(glycidyl methacrylate) (PGMA). Unlike uniform commercial PNIPAM coatings used for non-enzymatic cell detachment, these blended films phase-separate into PNIPAM matrices with embedded PS or PGMA domains whose size and spatial distribution are tunable by adjusting polymer concentration ratios. We hypothesize that thermally induced swelling of PNIPAM below its lower critical solution temperature (32 °C) generates surface fluctuations that selectively detach weakly adherent cells while strongly adherent cells remain bound to the hydrophobic domains. Combined with applied shear flow, this mechanism provides a basis for sorting cell populations by their differential adhesion strength.

Preliminary experiments identified optimal domain size, distribution, and PNIPAM film thickness for supporting cell attachment. We measured shear detachment forces across three cell types with distinct adhesion profiles and demonstrated proof-of-concept sorting. These results establish PNIPAM-based MS surfaces as a promising, antibody-free platform for cell sorting with potential applications in stem cell isolation and transplantation medicine.