Construction of 3-dimensional (3-D) engineered tissue is increasingly being investigated for use in drug discovery and regenerative medicine. Here, we developed multi-layered 3-D cellular assembly by using magnetic nanoparticles (MNP) isolated from Magnetospirillum sp. AMB-1 magnetotactic bacteria. Magnetized human dermal fibroblasts (HDFBs) were prepared by treatment with the MNP, induced to form 3-D assembly under a magnetic field. Analyses including LIVE/DEAD assay, transmission electron microscopy revealed that the MNP were internalized via clathrin-mediated endocytosis without cytotoxicity. The magnetized HDFBs could build 3-D structure as a function of seeding density. When the highest seeding density (5×105 cells/mm2) was used, the thickness of assembly was 41.90±1.69μm, with approximately 9.3±1.6 cell layers being formed. Immunofluorescence staining confirmed homogeneous distribution of ECM and junction proteins throughout the 3-D assembly. Real-time PCR analysis showed decrease in expression levels of collagen types I and IV but increase in that of connexin 43 in the 3-D assembly compared with the 2-D culture. Finally, we demonstrated that the discernible layers can be formed hierarchically by serial assembly. In conclusion, our study showed that a multi-layered structure can be easily prepared using magnetically-assisted cellular assembly with highlighting cell-cell and cell-ECM communication.
Bibliographical noteFunding Information:
This work was supported by a National Research Foundation of Korea (NRF) funded by the the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A03067809) and Radiation Technology R&D program (NRF-2015M2A2A6021052).
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- 3-D Cellular Assembly
- Cellular Internalization
- Hierarchical Tissue Mimicry
- Magnetic Nanoparticles
- Multi-Layered Structure