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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-based live/dead viability assay of one-layered, BMP2-immobilized C2C12 cell sheets (1-CS w/BMP2i) after 1-d (left) or 7-d (right) cultivation. (B) Relative cell viability of one-layered (1-CS w/BMP2i) and three-layered (3-CS w/BMP2i) BMP2-immobilized cell sheet(s) after 1-d or 7-d cultivation.
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Figure 4: (A) Fluorescence-based live/dead viability assay of one-layered, BMP2-immobilized C2C12 cell sheets (1-CS w/BMP2i) after 1-d (left) or 7-d (right) cultivation. (B) Relative cell viability of one-layered (1-CS w/BMP2i) and three-layered (3-CS w/BMP2i) BMP2-immobilized cell sheet(s) after 1-d or 7-d cultivation.

Mentions: We also investigated the viability of monolayered or multilayered cell sheet(s) after 1 d and 7 d of incubation, and we found that C2C12 cells remained healthy even after 7 d (Figure 4A-B). In addition, the morphology of a monolayer of cells was investigated using phase contrast and fluorescence microscopy (Figure 5A-F). In BMP2-labeled C2C12 cell sheets, cells were round in shape and exhibited an osteogenic phenotype after only 4 d, which was a pattern comparable to that of cells from culture media where an equivalent amount of BMP2 had been added. In contrast, cells cultured in a conventional culture dish without BMP2 exhibited a normal spindle-shaped morphology. We also used confocal laser scanning microscopy to observe a bilayer composed of C2C12 cells (Figure 5G). After sequential electrical stimulation, the recovered second cell layer overlapped with the first cell layer, thereby mimicking 3D tissue formation. The fabricated cell sheets had an approximate thickness of 35 µm. We explored the effect of BMP2-immobilized C2C12 cell sheets on osteoblastic expression. After electrical stimulation, BMP2-immobilized cell sheets (CS w/BMP2i) were transferred to a culture dish to assess alkaline phosphatase (ALP) activity in the C2C12 cells (Figure 6A). ALP activity in single-layered BMP2-immobilized cell sheets (1-CS w/BMP2i) apparently improved 4-fold compared with that in C2C12 cells cultured in BMP2-free media (CS w/o BMP2a). Additionally, ALP activity was significantly higher in multilayered sheets than single layers, which indicated that 3D tissues created by stacking individual cell sheets could be more therapeutically effective when transplanted. In comparison with cell sheets cultured in BMP2-added media (CS w/BMP2a), the enhanced ALP activity in CS w/BMP2i could be attributed to membrane-bound growth factors, which achieve receptor-ligand complex formation. Growth factor-tethered cell sheets showed efficient cellular activity that was significantly correlated with the defined amount of BMP2 present in individual cells, which would be unlikely with traditional soluble delivery methods. Indeed, direct binding of biomolecules to individual cells yields uniform distribution and long-term contact duration by restricting diffusion from the integration site, and thereby efficiently triggers differentiation. We also examined osteoblastic differentiation of C2C12 cells within 3D cell constructs by using an Alizarin red staining assay (Figure 6B-C). Interestingly, CS w/BMP2i showed more intense red staining than CS w/BMP2a in both normal cell media and osteogenic media. The results of Alizarin red staining suggested that BMP2-bound cell sheets favorably affect the induction of osteogenic differentiation and increase the accumulation of mineralized calcium phosphate. We found that CS w/BMP2i induced a 4-fold increase in mineral deposition compared to CS w/BMP2a, especially in osteogenic media.


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

Lee H, Cho Y - Theranostics (2015)

(A) Fluorescence-based live/dead viability assay of one-layered, BMP2-immobilized C2C12 cell sheets (1-CS w/BMP2i) after 1-d (left) or 7-d (right) cultivation. (B) Relative cell viability of one-layered (1-CS w/BMP2i) and three-layered (3-CS w/BMP2i) BMP2-immobilized cell sheet(s) after 1-d or 7-d cultivation.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: (A) Fluorescence-based live/dead viability assay of one-layered, BMP2-immobilized C2C12 cell sheets (1-CS w/BMP2i) after 1-d (left) or 7-d (right) cultivation. (B) Relative cell viability of one-layered (1-CS w/BMP2i) and three-layered (3-CS w/BMP2i) BMP2-immobilized cell sheet(s) after 1-d or 7-d cultivation.
Mentions: We also investigated the viability of monolayered or multilayered cell sheet(s) after 1 d and 7 d of incubation, and we found that C2C12 cells remained healthy even after 7 d (Figure 4A-B). In addition, the morphology of a monolayer of cells was investigated using phase contrast and fluorescence microscopy (Figure 5A-F). In BMP2-labeled C2C12 cell sheets, cells were round in shape and exhibited an osteogenic phenotype after only 4 d, which was a pattern comparable to that of cells from culture media where an equivalent amount of BMP2 had been added. In contrast, cells cultured in a conventional culture dish without BMP2 exhibited a normal spindle-shaped morphology. We also used confocal laser scanning microscopy to observe a bilayer composed of C2C12 cells (Figure 5G). After sequential electrical stimulation, the recovered second cell layer overlapped with the first cell layer, thereby mimicking 3D tissue formation. The fabricated cell sheets had an approximate thickness of 35 µm. We explored the effect of BMP2-immobilized C2C12 cell sheets on osteoblastic expression. After electrical stimulation, BMP2-immobilized cell sheets (CS w/BMP2i) were transferred to a culture dish to assess alkaline phosphatase (ALP) activity in the C2C12 cells (Figure 6A). ALP activity in single-layered BMP2-immobilized cell sheets (1-CS w/BMP2i) apparently improved 4-fold compared with that in C2C12 cells cultured in BMP2-free media (CS w/o BMP2a). Additionally, ALP activity was significantly higher in multilayered sheets than single layers, which indicated that 3D tissues created by stacking individual cell sheets could be more therapeutically effective when transplanted. In comparison with cell sheets cultured in BMP2-added media (CS w/BMP2a), the enhanced ALP activity in CS w/BMP2i could be attributed to membrane-bound growth factors, which achieve receptor-ligand complex formation. Growth factor-tethered cell sheets showed efficient cellular activity that was significantly correlated with the defined amount of BMP2 present in individual cells, which would be unlikely with traditional soluble delivery methods. Indeed, direct binding of biomolecules to individual cells yields uniform distribution and long-term contact duration by restricting diffusion from the integration site, and thereby efficiently triggers differentiation. We also examined osteoblastic differentiation of C2C12 cells within 3D cell constructs by using an Alizarin red staining assay (Figure 6B-C). Interestingly, CS w/BMP2i showed more intense red staining than CS w/BMP2a in both normal cell media and osteogenic media. The results of Alizarin red staining suggested that BMP2-bound cell sheets favorably affect the induction of osteogenic differentiation and increase the accumulation of mineralized calcium phosphate. We found that CS w/BMP2i induced a 4-fold increase in mineral deposition compared to CS w/BMP2a, especially in osteogenic media.

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.