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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.


(A) Fluorescence microscopy images of polypyrrole (Ppy) only (top), and bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy before (middle) and after (bottom) electrical stimulation. Electrical potential (-0.8 V for 30 s) induced massive release of biotin and conjugated BMP2 from Ppy. Surface-immobilized BMP2 was visualized using fluorescein isothiocyanate (FITC)-conjugated anti-BMP2 antibody. (B) Quantification (by ELISA) of BMP2 immobilized on biotin-doped Ppy surfaces by using various protein concentrations. (C) Fluorescence images of BMP2-conjugated C2C12 cells (upper panels) and normal C2C12 cells (lower panels). After electrical stimulation, the detached BMP2-conjugated cells were incubated in a solution containing anti-BMP2 labeled FITC.
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Figure 2: (A) Fluorescence microscopy images of polypyrrole (Ppy) only (top), and bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy before (middle) and after (bottom) electrical stimulation. Electrical potential (-0.8 V for 30 s) induced massive release of biotin and conjugated BMP2 from Ppy. Surface-immobilized BMP2 was visualized using fluorescein isothiocyanate (FITC)-conjugated anti-BMP2 antibody. (B) Quantification (by ELISA) of BMP2 immobilized on biotin-doped Ppy surfaces by using various protein concentrations. (C) Fluorescence images of BMP2-conjugated C2C12 cells (upper panels) and normal C2C12 cells (lower panels). After electrical stimulation, the detached BMP2-conjugated cells were incubated in a solution containing anti-BMP2 labeled FITC.

Mentions: Here, we explored the feasibility of this approach by investigating the efficacy of immobilization of biotinylated BMP2 to the functionalized Ppy surface. In general, C2C12 cells are cultured in medium containing BMP2. Under these circumstances, soluble delivery of proteins often restricts their availability to cell surface receptors. We attempted to overcome this problem by constructing cell sheets with BMP2 ligands tagged to individual cells. Initially, we analyzed the binding efficacy of BMP2 to biotin-doped Ppy by using fluorescence microscopy; fluorescence images were used to examine the distribution of surface-immobilized proteins, with fluorescein isothiocyanate (FITC)-conjugated anti-BMP2 employed as a detection probe (Figure 2A). The green fluorescent regions visualized on Ppy surfaces indicated the existence of BMP2 because of the preferential association of the biotin-streptavidin linkage. However, in response to an applied electric field, the fluorescent signals disappeared from the electrically stimulated Ppy surface, which can primarily be explained by the massive release of biotin and conjugated BMP2 from Ppy. We also examined the BMP2 loading efficacy by using various concentrations. As shown in Figure 2B, when 50-300 ng/cm2 BMP2 was applied onto the biotin-doped Ppy surfaces, the amount of immobilized BMP2 (quantified by ELISA) was 39-147 ng/cm2, suggesting that the levels of surface-immobilized BMP2 would be sufficient to enhance its availability to cell surface receptors. In addition, we demonstrated the presence of BMP2 on the surface of C2C12 cells (Figure 2C). By applying an electric field, we released cells specifically bound with growth factors to membrane receptors. BMP2-conjugated C2C12 cells were then incubated in a solution containing anti-BMP2 labeled with FITC. Finally, BMP2 was observed along the cell membranes of BMP2-conjugated C2C12 cells but not in normal C2C12 cells (Figure 2C). In the present study, we proposed that individual cells within a cell sheet could be labeled with BMP2 to stimulate osteogenesis in C2C12 myogenic cells. Because the biotin within the Ppy surface allows greater flexibility for the incorporation of new biological moieties, this could be a versatile and molecularly well-defined methodology for cell surface engineering and may be compatible with a variety of cellular applications.


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

Lee H, Cho Y - Theranostics (2015)

(A) Fluorescence microscopy images of polypyrrole (Ppy) only (top), and bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy before (middle) and after (bottom) electrical stimulation. Electrical potential (-0.8 V for 30 s) induced massive release of biotin and conjugated BMP2 from Ppy. Surface-immobilized BMP2 was visualized using fluorescein isothiocyanate (FITC)-conjugated anti-BMP2 antibody. (B) Quantification (by ELISA) of BMP2 immobilized on biotin-doped Ppy surfaces by using various protein concentrations. (C) Fluorescence images of BMP2-conjugated C2C12 cells (upper panels) and normal C2C12 cells (lower panels). After electrical stimulation, the detached BMP2-conjugated cells were incubated in a solution containing anti-BMP2 labeled FITC.
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Related In: Results  -  Collection

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Figure 2: (A) Fluorescence microscopy images of polypyrrole (Ppy) only (top), and bone morphogenetic protein 2 (BMP2)-immobilized biotin-doped Ppy before (middle) and after (bottom) electrical stimulation. Electrical potential (-0.8 V for 30 s) induced massive release of biotin and conjugated BMP2 from Ppy. Surface-immobilized BMP2 was visualized using fluorescein isothiocyanate (FITC)-conjugated anti-BMP2 antibody. (B) Quantification (by ELISA) of BMP2 immobilized on biotin-doped Ppy surfaces by using various protein concentrations. (C) Fluorescence images of BMP2-conjugated C2C12 cells (upper panels) and normal C2C12 cells (lower panels). After electrical stimulation, the detached BMP2-conjugated cells were incubated in a solution containing anti-BMP2 labeled FITC.
Mentions: Here, we explored the feasibility of this approach by investigating the efficacy of immobilization of biotinylated BMP2 to the functionalized Ppy surface. In general, C2C12 cells are cultured in medium containing BMP2. Under these circumstances, soluble delivery of proteins often restricts their availability to cell surface receptors. We attempted to overcome this problem by constructing cell sheets with BMP2 ligands tagged to individual cells. Initially, we analyzed the binding efficacy of BMP2 to biotin-doped Ppy by using fluorescence microscopy; fluorescence images were used to examine the distribution of surface-immobilized proteins, with fluorescein isothiocyanate (FITC)-conjugated anti-BMP2 employed as a detection probe (Figure 2A). The green fluorescent regions visualized on Ppy surfaces indicated the existence of BMP2 because of the preferential association of the biotin-streptavidin linkage. However, in response to an applied electric field, the fluorescent signals disappeared from the electrically stimulated Ppy surface, which can primarily be explained by the massive release of biotin and conjugated BMP2 from Ppy. We also examined the BMP2 loading efficacy by using various concentrations. As shown in Figure 2B, when 50-300 ng/cm2 BMP2 was applied onto the biotin-doped Ppy surfaces, the amount of immobilized BMP2 (quantified by ELISA) was 39-147 ng/cm2, suggesting that the levels of surface-immobilized BMP2 would be sufficient to enhance its availability to cell surface receptors. In addition, we demonstrated the presence of BMP2 on the surface of C2C12 cells (Figure 2C). By applying an electric field, we released cells specifically bound with growth factors to membrane receptors. BMP2-conjugated C2C12 cells were then incubated in a solution containing anti-BMP2 labeled with FITC. Finally, BMP2 was observed along the cell membranes of BMP2-conjugated C2C12 cells but not in normal C2C12 cells (Figure 2C). In the present study, we proposed that individual cells within a cell sheet could be labeled with BMP2 to stimulate osteogenesis in C2C12 myogenic cells. Because the biotin within the Ppy surface allows greater flexibility for the incorporation of new biological moieties, this could be a versatile and molecularly well-defined methodology for cell surface engineering and may be compatible with a variety of cellular applications.

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.