Human Amniotic Cell Sheet Harvest Using a Novel Temperature-Responsive Culture Surface Coated with Protein-Based Polymer

Abstract

Human amniotic epithelial (hAE) and mesenchymal (hAM) cells are believed to have the potential to differentiate into various functional cells, such as neurons, hepatocytes, cardiomyocytes, and pancreatic beta cells. However, cell transplantation has been performed by injection of cell suspensions, and thus it is difficult to control shape, size, location, and functions of differentiated cells. To overcome these problems, we developed a novel temperature-responsive culture surface coated with elastic protein-based polymer. By reducing the temperature using a polyvinylidene difluoride (PVDF) membrane, the primary hAE and hAM cell sheet can detach from the coated surface. The recovered cell sheet can be transferred and can re-adhere and re-proliferate on another surface. This represents the first report of harvesting of primary hAE and hAM cell sheets using the novel temperature- responsive polymer. These findings suggest that this new technique of cell sheet detachment from noncytotoxic, highly biocompatible protein-based polymer-coated surfaces may be useful in tissue engineering, as well as in the investigation of hAE and hAM cell sheets for transplantation.

Peptide-based biopolymers in biomedicine and biotechnology

“…Examples can be found of ELP hydrogels that are formed by irradiation [246][247][248], photoinitiation [249], amine-reactive chemical crosslinking [37,68,[250][251][252][253][254][255][256][257][258][259][260], and enzymatic crosslinking by tissue transglutaminase [261]. The hydrogels have been successfully used for cartilage and intervertebral disc tissue repair, small-diameter vascular grafts, urinary bladders, stem cell matrices, neural guides, stem cell sheets, and post-surgical wound treatment [250][251][252][253][254][255][256][262][263][264]. McMillan et al used the electrophilic crosslinker bis(sulfosuccinimidyl) suberate to join lysine residues in a lysine-rich ELP to form hydrogels in either phosphate buffer at pH 8.5 or anhydrous dimethylsulfoxide.…”

Engineering the Extracellular Environment: Strategies for Building 2D and 3D Cellular Structures

“…[ 211 ] Besides PNIPAAm and its derivatives, other temperature-responsive polymers have been successfully applied for cell sheet engineering. A highly biocompatible protein-based thermo-sensitive polymer, containing fi bronectin and elastin precursor sequences showed an inverse temperature transition of 29 ° C, and allowed cell sheet detachment at 4 ° C. [ 212 ] Similarly, a thermo-reversible salts-blended methylcellulose hydrogel coated with a thin collagen layer allowed for cell sheet recovery at 20 ° C, while being applicable to any surfaces by simple solution coating. [ 213 ] To envisage the biofabrication of functional organ-like structures, strategies for the co-culture of different cell types, with controlled spatial distribution and orientation, are necessary.…”

Elastin‐Like Macromolecules

“…By reducing the temperature using a polyvinylidene difluoride (PVDF) membrane, the cell sheet was detached from the coated surface and subsequently transferred to new surfaces, suggesting its potential for the fabrication of multilayer cell sheets for transplantation [24]. For bone tissue engineering, an ELR-RGD has been adsorbed onto micropatterned poly(N-isopropyl acrylamide) films displaying a positive maintenance of the cell attachment in the scaffolds under dynamic culture conditions [168].…”