Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer

Abstract

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.

Novel temperature-responsive polymer brushes with carbohydrate residues facilitate selective adhesion and collection of hepatocytes

“…PNIPAAm surfaces that reversibly bind and release proteins and cells have been developed for analytical and biomedical applications [8][9][10][11]. Anchor-dependent cells, for example, can adhere and spread well on the PNIPAAm brush surfaces at 37…”

“…• C (above the lower critical solution temperature; LCST), and intact cells can be collected from the surfaces below the LCST [8]. Because this method can avoid the use of deleterious enzymes, cells are harvested as a single contiguous sheet after they become confluent [12].…”

On the Interaction of Adherent Cells with Thermoresponsive Polymer Coatings

“…Moreover, this argument is in line with results obtained by colloidal probe measurements which suggest that the kinetics of the change of cell morphology upon the decrease of temperature is determined by cellular processes rather than by the dynamics of the transition of the polymers [20]. The assumption of an actively driven cell detachment on thermoresponsive surfaces is further supported by observations of Okano et al [21] on poly(N-isopropylacrylamide)-coatings who also described a reduced cell detachment efficiency at lower temperatures and after chemical manipulation of the cell metabolism [22,23]. A phenomenon frequently observed when the migration or rounding of adherent cells on synthetic surfaces is monitored is that the cells leave residues behind on the surface from where they detach.…”

Tissue Engineering of the Corneal Endothelium: A Review of Carrier Materials

“…The mechanism of adhesion and detachment of cells to or from thermo-responsive cell culture carriers has been widely investigated [128,129,130,131,132]. The adhesion of cells above the T cr (hydrophobic state) is a complex combination of physicochemical effects, like hydrophobic and electrostatic interactions, as well as van der Waals forces.…”