Cell-adhesive and cell-repulsive zwitterionic oligopeptides for micropatterning and rapid electrochemical detachment of cells

Abstract

In this study, we describe the development of oligopeptide-modified cell culture surfaces from which adherent cells can be rapidly detached by application of an electrical stimulus. An oligopeptide, CGGGKEKEKEK, was designed with a terminal cysteine residue to mediate binding to a gold surface via a gold-thiolate bond. The peptide forms a self-assembled monolayer through the electrostatic force between the sequence of alternating charged glutamic acid (E) and lysine (K) residues. The dense and electrically neutral oligopeptide zwitterionic layer of the modified surface was resistant to nonspecific adsorption of proteins and adhesion of cells, while the surface was altered to cell adhesive by the addition of a second oligopeptide (CGGGKEKEKEKGRGDSP) containing the RGD cell adhesion motif. Application of a negative electrical potential to this gold surface cleaved the gold-thiolate bond, leading to desorption of the oligopeptide layer, and rapid (within 2 min) detachment of virtually all cells. This approach was applicable not only to detachment of cell sheets but also for transfer of cell micropatterns to a hydrogel. This electrochemical approach of cell detachment may be a useful tool for tissue-engineering applications.

FIG. 1.

Design of zwitterionic oligopeptides. (A) Cell-repulsive oligopeptide, CGGGKEKEKEK. (B) Cell-adhesive oligopeptide, CGGGKEKEKEKGRGDSP. Yellow, cysteine; white, glycine; blue, lysine; and red, glutamic acid. Both oligopeptides contain a terminal cysteine C that mediates binding to the gold surface through a gold–thiolate bond. Oligopeptides also contain a linker of three glycines G and an alternating positively charged lysine K and negatively charged glutamic acid E residues. K and E support formation of a dense self-assembled monolayer (SAM) through electrostatic forces. (C) SAM of the cell-repulsive oligopeptide. (D) SAM of the mixture of cell-adhesive and cell-repulsive oligopeptide. Color images available online at www.liebertpub.com/tea

FIG. 2.

Oligopeptide-modified gold surfaces and their nonfouling properties. (A) Spontaneous adsorption of cell-repulsive, cell-adhesive, and bridge-shaped oligopeptides. (B) Fibronectin and fibrinogen adsorption onto bare gold surfaces, or gold surfaces modified with the bridge-shaped or cell-repulsive oligopeptides. The error bars indicate SD calculated from three independent quartz crystal microbalance measurements. *p<0.05 compared to the bare gold and bridge-shaped oligopeptide surfaces.

FIG. 3.

Cell adhesion and growth on substrates prepared with different ratios of zwitterionic oligopeptides. (A) Phase-contrast images of cells after 3 h of culture. The percentage value above each panel indicates the molar ratio of the cell-adhesive oligopeptide to the cell-repulsive oligopeptide. (B) Quantitation of adherent cells obtained from image analyses. (C) Growth of adherent cells. The error bars indicate SD calculated from three independent experiments for each substrate. *p<0.05 compared to the 100% cell-adhesive oligopeptide (B), or equal to or less than 0.1% cell-adhesive oligopeptide (C). Color images available online at www.liebertpub.com/tea

FIG. 4.

Cell detachment after electrochemical desorption of the zwitterionic oligopeptides. (A) Cyclic voltammogram obtained during the reductive desorption of the oligopeptide; 1, 2, and 3 are scan numbers. Cyclic voltammograms were recorded at a scanning rate of 20 mV/s with respect to an Ag/AgCl reference electrode. The working electrode area was 8.0 mm². (B) Schematic diagram of the electrochemical detachment of cells along with desorption of the oligopeptide. (C) Phase-contrast micrographs indicating fibroblasts on the gold surface modified with the cell-adhesive oligopeptide were readily detached within 2 min. (D) Percentage fibroblasts remaining bound to substrate after negative potential application. Cells were enumerated by image analysis of surfaces modified with the cell-adhesive oligopeptide, bridge-shaped oligopeptide, 10-carboxy-1-decanethiol, or bare gold surface. Potential application was −1.0 V with respect to an Ag/AgCl reference. The error bars indicate SD calculated from three independent experiments for each plot. *p<0.05 compared to the bridge-shaped oligopeptide. Color images available online at www.liebertpub.com/tea

FIG. 5.

Electrochemical transfer of cell sheets and micropatterned cells to collagen gels. (A) Detached human bone marrow-derived mesenchymal stem cell (hBMSC) sheet. (B) Live/dead fluorescent staining of the detached hBMSC sheets: green indicates live cells, and red indicates dead cells. (C) Procedure for micropatterning of the two oligopeptides. (D) Stamped fluorescein patterning. Fibroblasts adherent on the micropatterned surface at 3 h (E), 1 day (F), and 2 days (G) of culture and on striped pattern at 1 day (H) and 2 days (I) of culture. (J) Human umbilical vein endothelial cells constitutively expressing green fluorescent protein (GFP-HUVECs) on striped micropatterns at 3 h of culture. GFP-HUVECs transferred to collagen gels and cultured for 1 (K), 2 (L), and 3 days (M). Color images available online at www.liebertpub.com/tea