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An Innovative Strategy for the Fabrication of Functional Cell Sheets Using an Electroactive Conducting Polymer.

Lee H, Cho Y - Theranostics (2015)

Bottom Line: Here, we report the development of an electric field-assisted methodology for constructing 3D C2C12 cell sheets with the potential for cell surface modification.Subsequently, C2C12 cells are cultured on BMP2-immobilized Ppy surfaces to induce interactions between cell surface receptors and bound BMP2 ligands.Following these procedures, layers of BMP2-immobilized cells can be easily detached from the Ppy surface by applying an electrical potential.

View Article: PubMed Central - PubMed

Affiliation: New Experimental Therapeutic Branch, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang, Gyeonggi-do 410-769, South Korea.

ABSTRACT
Here, we report the development of an electric field-assisted methodology for constructing 3D C2C12 cell sheets with the potential for cell surface modification. In this method, a conducting polymer, polypyrrole (Ppy), is electrodeposited via biotin doping, and then chemical conjugation of biotinylated bone morphogenetic protein 2 (BMP2) is achieved using a biotin-streptavidin cross-linker. Subsequently, C2C12 cells are cultured on BMP2-immobilized Ppy surfaces to induce interactions between cell surface receptors and bound BMP2 ligands. Following these procedures, layers of BMP2-immobilized cells can be easily detached from the Ppy surface by applying an electrical potential. This novel method results in high affinity, ligand-bound cell sheets, which exhibit homogeneous coverage with membrane-bound proteins and signal activation that occurs via maximal receptor accessibility. Using this strategy to engineer the cell surface with desirable ligands results in structures that mimic in vivo tissues; thus, the method reported here has potential applications in regenerative medicine and tissue engineering.

No MeSH data available.


Related in: MedlinePlus

(A) Bright-field photography of C2C12 cell sheet(s) detached from biotin-doped polypyrrole (Ppy) in response to electrical stimulation (-0.8 V for 30 s). Inset image shows Ppy surface after the detachment of the cell sheet. 3D cell sheets were fabricated by repeatedly layering detached cell sheets in a 35-mm cell culture dish. (B) Efficiency of cell detachment from bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy in response to electric fields (+0.4 V to -0.8 V for 30 s). (C) Effect of electrical stimulation on the cyclic voltammogram curve, with a 5-mM ferricyanide probe solution used as an indicator.
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Figure 3: (A) Bright-field photography of C2C12 cell sheet(s) detached from biotin-doped polypyrrole (Ppy) in response to electrical stimulation (-0.8 V for 30 s). Inset image shows Ppy surface after the detachment of the cell sheet. 3D cell sheets were fabricated by repeatedly layering detached cell sheets in a 35-mm cell culture dish. (B) Efficiency of cell detachment from bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy in response to electric fields (+0.4 V to -0.8 V for 30 s). (C) Effect of electrical stimulation on the cyclic voltammogram curve, with a 5-mM ferricyanide probe solution used as an indicator.

Mentions: As shown in Figure 3, we also demonstrated the construction of BMP2-conjugated C2C12 cell sheets. First, C2C12 cells (5 × 104) were seeded on a BMP2-immobilized biotin-doped Ppy surface for 5 d to facilitate a tight interface between cells. The cells assembled as a sheet could be non-destructively detached from the Ppy surface by applying electric fields (Figure 3A). Indeed, a weak electric potential delicately modulated the unique cell-surface interface by causing the spontaneous release of biotin moieties and attached cells from the surface. Recovered cell sheets could be easily fabricated as 3D cellular multilayers. We also investigated the effect of electric potentials on manipulating cell sheet technology by applying electrical fields ranging from -0.8 V to +0.4 V to the Ppy surfaces for 30 s (Figure 3B). Consistent with our previous studies,18, 19 the monolayered cell sheet was readily detached from the surface by gentle agitation with a PBS-filled pipette following negative electrical stimulation. However, electrically positive stimulation of Ppy surfaces produced no reaction, with results that were similar to those of the unstimulated control. We further evaluated the electrochemical behavior of Ppy surfaces by using cyclic voltammetry (CV), as shown in Figure 3C. Redox peak currents were enhanced on the biotin-doped Ppy surface. However, C2C12 cell sheets grown on BMP2-immobilized, biotin-doped Ppy surfaces exhibited obviously decreased electron-transfer capabilities, specifically of electroactive ferricyanide species throughout the Ppy surfaces, because of tight cell-surface junctions. Electrical stimulation at -0.8 V was sufficient to detach the cell sheet from the Ppy surface, which could ultimately restore the current intensity to its original state by allowing the free transfer of electrolytes. We noted, however, that a positive potential did not affect peak intensity, which indicates that C2C12 cell sheets remained firmly attached to the Ppy surface regardless of the applied electrical stimulation. Electrical stimulation induces the conformational change of Ppy backbones via oxidation/reduction reactions; specifically, the Ppy polymer swells significantly with a positive potential, generating free volume and enabling entrapment of various moieties inside the polymeric backbone.23 In contrast, at negative potentials, Ppy undergoes structural shrinkage and squeezes out molecules incorporated within the polymer. Indeed, our results demonstrated preferential detachment of cell sheets only when negative electrical potentials were applied.


An Innovative Strategy for the Fabrication of Functional Cell Sheets Using an Electroactive Conducting Polymer.

Lee H, Cho Y - Theranostics (2015)

(A) Bright-field photography of C2C12 cell sheet(s) detached from biotin-doped polypyrrole (Ppy) in response to electrical stimulation (-0.8 V for 30 s). Inset image shows Ppy surface after the detachment of the cell sheet. 3D cell sheets were fabricated by repeatedly layering detached cell sheets in a 35-mm cell culture dish. (B) Efficiency of cell detachment from bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy in response to electric fields (+0.4 V to -0.8 V for 30 s). (C) Effect of electrical stimulation on the cyclic voltammogram curve, with a 5-mM ferricyanide probe solution used as an indicator.
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Related In: Results  -  Collection

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Figure 3: (A) Bright-field photography of C2C12 cell sheet(s) detached from biotin-doped polypyrrole (Ppy) in response to electrical stimulation (-0.8 V for 30 s). Inset image shows Ppy surface after the detachment of the cell sheet. 3D cell sheets were fabricated by repeatedly layering detached cell sheets in a 35-mm cell culture dish. (B) Efficiency of cell detachment from bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy in response to electric fields (+0.4 V to -0.8 V for 30 s). (C) Effect of electrical stimulation on the cyclic voltammogram curve, with a 5-mM ferricyanide probe solution used as an indicator.
Mentions: As shown in Figure 3, we also demonstrated the construction of BMP2-conjugated C2C12 cell sheets. First, C2C12 cells (5 × 104) were seeded on a BMP2-immobilized biotin-doped Ppy surface for 5 d to facilitate a tight interface between cells. The cells assembled as a sheet could be non-destructively detached from the Ppy surface by applying electric fields (Figure 3A). Indeed, a weak electric potential delicately modulated the unique cell-surface interface by causing the spontaneous release of biotin moieties and attached cells from the surface. Recovered cell sheets could be easily fabricated as 3D cellular multilayers. We also investigated the effect of electric potentials on manipulating cell sheet technology by applying electrical fields ranging from -0.8 V to +0.4 V to the Ppy surfaces for 30 s (Figure 3B). Consistent with our previous studies,18, 19 the monolayered cell sheet was readily detached from the surface by gentle agitation with a PBS-filled pipette following negative electrical stimulation. However, electrically positive stimulation of Ppy surfaces produced no reaction, with results that were similar to those of the unstimulated control. We further evaluated the electrochemical behavior of Ppy surfaces by using cyclic voltammetry (CV), as shown in Figure 3C. Redox peak currents were enhanced on the biotin-doped Ppy surface. However, C2C12 cell sheets grown on BMP2-immobilized, biotin-doped Ppy surfaces exhibited obviously decreased electron-transfer capabilities, specifically of electroactive ferricyanide species throughout the Ppy surfaces, because of tight cell-surface junctions. Electrical stimulation at -0.8 V was sufficient to detach the cell sheet from the Ppy surface, which could ultimately restore the current intensity to its original state by allowing the free transfer of electrolytes. We noted, however, that a positive potential did not affect peak intensity, which indicates that C2C12 cell sheets remained firmly attached to the Ppy surface regardless of the applied electrical stimulation. Electrical stimulation induces the conformational change of Ppy backbones via oxidation/reduction reactions; specifically, the Ppy polymer swells significantly with a positive potential, generating free volume and enabling entrapment of various moieties inside the polymeric backbone.23 In contrast, at negative potentials, Ppy undergoes structural shrinkage and squeezes out molecules incorporated within the polymer. Indeed, our results demonstrated preferential detachment of cell sheets only when negative electrical potentials were applied.

Bottom Line: Here, we report the development of an electric field-assisted methodology for constructing 3D C2C12 cell sheets with the potential for cell surface modification.Subsequently, C2C12 cells are cultured on BMP2-immobilized Ppy surfaces to induce interactions between cell surface receptors and bound BMP2 ligands.Following these procedures, layers of BMP2-immobilized cells can be easily detached from the Ppy surface by applying an electrical potential.

View Article: PubMed Central - PubMed

Affiliation: New Experimental Therapeutic Branch, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang, Gyeonggi-do 410-769, South Korea.

ABSTRACT
Here, we report the development of an electric field-assisted methodology for constructing 3D C2C12 cell sheets with the potential for cell surface modification. In this method, a conducting polymer, polypyrrole (Ppy), is electrodeposited via biotin doping, and then chemical conjugation of biotinylated bone morphogenetic protein 2 (BMP2) is achieved using a biotin-streptavidin cross-linker. Subsequently, C2C12 cells are cultured on BMP2-immobilized Ppy surfaces to induce interactions between cell surface receptors and bound BMP2 ligands. Following these procedures, layers of BMP2-immobilized cells can be easily detached from the Ppy surface by applying an electrical potential. This novel method results in high affinity, ligand-bound cell sheets, which exhibit homogeneous coverage with membrane-bound proteins and signal activation that occurs via maximal receptor accessibility. Using this strategy to engineer the cell surface with desirable ligands results in structures that mimic in vivo tissues; thus, the method reported here has potential applications in regenerative medicine and tissue engineering.

No MeSH data available.


Related in: MedlinePlus