Toward Label-Free Selective Cell Separation of Different Eukaryotic Cell Lines Using Thermoresponsive Homopolymer Layers

Abstract

Ultrathin thermoresponsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) homopolymer layers are reported as a novel platform for label-free temperature-stimulated cell separation from a coculture of eukaryotic cell lines. Pancreatic tumor cells (PaTu 8988t) and fibroblasts (NIH 3T3) were shown to attach and proliferate on PDEGMA layers with a dry thickness of 5 ± 1 nm at 37 °C. After the cell medium cooled to below the lower critical solution temperature (LCST), PaTu 8988t cells showed a significantly decreased cell area. By contrast, there was no significant change in cell area for NIH 3T3 cells, while they still exhibited lamellipodia and filopodia. PaTu 8988t cells could be detached at 22 °C after flushing gently with the cell medium, while negligible cell detachment was observed for NIH 3T3 cells under identical conditions. This significant difference between PaTu 8988t and NIH 3T3 was due to the different cell adhesion on the culture-medium-derived protein covering the PDEGMA layer. Thus, cell separation from a coculture was achieved successfully by cooling the cell medium to 22 °C, resulting in the desorption of 95 ± 6% of PaTu 8988t cells, while the NIH 3T3 cells remained adherent. Our findings provide an effective, label-free, rapid, and simple approach, exploiting a thermoresponsive homopolymer layer to separate and enrich or isolate cells from mixed cell populations for basic biological research, medical diagnostics, tissue engineering, and cell therapy.

Tunable Adhesion of Different Cell Types Modulated by Thermoresponsive Polymer Brush Thickness

“…In addition, the formation of filopodia and lamellipodia, which are built up by bundles of F-actin network, plays key roles in the cellular adhesion process. − The SEM images show that the long filamentous filopodia and thin sheetlike lamellipodia of the L929 cells can be clearly observed at 37 °C, which is one more piece of evidence for the strong attachment of L929 cells on the PN 2 surface (see the Supporting Information). Intriguingly, similar phenomena were also observed on the thermoresponsive PDEGMA layer in an earlier study …”

“…These results, when combined, imply that it is possible to modulate tunable adhesion of different cell types simply by tailoring the thickness of PNIPAAm brushes. Intriguingly, similar adhesion/detachment of cells depending on brush thickness was observed in a recent study . Another thermoresponsive polymer brush, poly­(di­(ethylene glycol) methyl ether methacrylate) (PDEGMA), allowed the adhesion of PaTu 8988t cells at the thickness of 5 ± 1 nm when the temperature was above the apparent transition temperature (∼32 °C).…”

“…These unique features make PNIPAAm an ideal substrate for the harvest of cell sheets without using chelating agents and proteolytic enzymes to break down basal ECM . More recently, another thermoresponsive poly­(diethylene glycol methacrylate) brush was reported for temperature-stimulated cell separation, and the action mechanisms for the regulation of cell adhesion and detachment were examined with respect to thickness and concomitant polymer brushes …”

“…26 More recently, another thermoresponsive poly(diethylene glycol methacrylate) brush was reported for temperature-stimulated cell separation, and the action mechanisms for the regulation of cell adhesion and detachment were examined with respect to thickness and concomitant polymer brushes. 27 Previously, researchers reported that the adhesion behavior of cells was greatly governed by the thickness of the PNIPAAm layers. 23 However, the underlying action mechanisms of PNIPAAm thickness on cell adhesion were still unknown.…”

Geometrical Constraints of Poly(diethylene glycol methyl ether methacrylate) Brushes on Spherical Nanoparticles and Cylindrical Nanowires: Implications for Thermoresponsive Brushes on Nanoobjects

“…37 With a special focus on PDEGMA brushes itself, the effective thermoresponsive behavior, which is associated with the lower critical solution temperature (LCST), can be modulated in ultrathin coatings around the physiological temperature, depending on the brush thicknesses. 38 Particularly attractive are applications for selective cell release coatings, 39,40 which are potentially enhanced on curves nanomaterials. Only a limited number of studies have addressed the understanding of the polymer brush growth on regularly aligned SiNWs with adjusted interwire distances.…”