Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces

Abstract

Temperature-responsive hydration/dehydration changes in surface-grafted poly(N-isopropylacrylamide) (PIPAAm) were utilized for hydrophilic/hydrophobic surface property alterations in cell culture. In this report, we utilized PIPAAm-grafted surfaces to recover confluently-cultured vascular endothelial cells as coherent monolayers from this cell culture substrate and to transfer to new cell culture substrates. For this purpose, we used two different methods to recover and transfer cell monolayer cultures: (1) chitin membranes used as an apical side cell support during cultured cell transfer, allowing cell basal side reattachment to new culture substrates after transfer; and (2) a cell culture insert (porous PET) used as both a support as well as new substrate, allowing basal surfaces of cultured cells to be exposed to the medium after transfer. In both cases, all cells grown on PIPAAm-grafted surfaces detach completely with maintenance of basement membrane-like structure. Recovered cells attach to the second culture surfaces, covering more than 60% of the new substrate, and retain approximately 90% viability and their original function as judged from tissue-type plasminogen activator secretion. This technique could be utilized to prepare novel bioartificial organs as well as cell co-culture systems by multi-layering different cell types to mimic tissue structures for tissue engineering.

Progress in Myocardial Regeneration and Cell Transplantation

“…48,48 Using this approach, another method of producing cardiomyocyte sheets (Fig 11) was developed in my laboratory. 50 This new method uses poly- merized fibrin (fibrin glue) normally used in surgical operations.…”

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets

“…3,17 Briefly, IPAAm monomer in 2-propanol solution was spread onto polystyrene culture dishes (Falcon 3001). Then, the dishes were subjected to electron beam irradiation with an Area Beam Electron Processing System (Nisshin High Voltage, Kyoto, Japan).…”

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

“…Because this method can avoid the use of deleterious enzymes, cells are harvested as a single contiguous sheet after they become confluent [12]. These transplantable cell sheets retain cell-cell junctional proteins as well as an intact extracellular matrix underneath the cell sheets, and therefore, they have been stratified in vitro [13] and directly transplanted in vivo [14]. PNIPAAm can also be functionalized by chemical conjugation to biomolecules such as targeting ligands to yield a large and diverse family of polymer-biomolecule hybrid systems [15].…”