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

Abstract

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.

Graphene oxide coating facilitates the bioactivity of scaffold material for tissue engineering

“…We speculate that inhibitory biological effects are involved in a high concentration of GO. Cells attach to hydrophobic surfaces via their cell membrane 47,48) , but 1 wt% GO films provide a fairly hydrophilic surface. Lee et al have demonstrated that contact angle of GO film was lower than that of reduced GO film without functional groups 14) .…”

Temperature-dependent interfacial properties of hydrophobically end-modified poly(2-isopropyl-2-oxazoline)s assemblies at the air/water interface and on solid substrates

“…Commercial devices that rely on the sharpness, tunability, reversibility, and reliability of this transition are now available in areas as varied as chromatography [1,2], microfluidics [3], cell culture, and tissue engineering [4,5]. These achievements have spurred a renewed interest in fundamental studies of polymer self-assembly at the air/water (A/W) interface particularly in the case of amphiphilic diblock copolymers [6][7][8].…”

Cell Attachment–Detachment Control on Temperature-Responsive Thin Surfaces for Novel Tissue Engineering

“…33 Canavan et al investigated the location of laminin, fibronectin, and type I and type IV collagens after cell detachment from plasma polymerized PIPAAm. [5][6][7] Immunoassays revealed that most fibronectin and laminin detached together with the cell sheets after reducing temperature.…”