Construction and Harvest of Multilayered Keratinocyte Sheets Using Magnetite Nanoparticles and Magnetic Force

Abstract

Novel technologies to establish three-dimensional constructs are desired for tissue engineering. In the present study, magnetic force was used to construct multilayered keratinocyte sheets and harvest the sheets without enzymatic treatment. Our original magnetite cationic liposomes, which have a positive surface charge in order to improve adsorption, were taken up by human keratinocytes at a concentration of 33 pg of magnetite per cell. The magnetically labeled keratinocytes (2x10(6) cells, which corresponds to 5 times the confluent concentration against the culture area of 24-well plates, in order to produce 5-layered keratinocyte sheets) were seeded into a 24-well ultralow-attachment plate, the surface of which was composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. A magnet (4000 G) was placed under the well, and the keratinocytes formed a five-layered construct in low-calcium medium (calcium concentration, 0.15 mM) after 24 h of culture. Subsequently, when the five-layered keratinocytes were cultured in high-calcium medium (calcium concentration, 1.0 mM), keratinocytes further stratified, resulting in the formation of 10-layered epidermal sheets. When the magnet was removed, the sheets were detached from the bottom of the plates, and the sheets could be harvested with a magnet. These results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach for tissue engineering.

Incorporation of Capillary-Like Structures into Dermal Cell Sheets Constructed by Magnetic Force-Based Tissue Engineering

“…We previously reported that MCLs do not adversely affect proliferation of several types of normal cells within the range of MCL concentrations tested (i.e., human aortic endothelial cells [HAECs], <100 pg-magnetite/cell (Ito et al, 2004c); human keratinocytes, <50 pg/cell (Ito et al, 2004a); human smooth muscle cells, <100 pg-magnetite/cell (Ito et al, 2005b); human mesenchymal stem cells [MSCs], <100 pg/cell (Ito et al, 2004b)), and we observed no effects on MSC differentiation (Ito et al, 2004b). However, further study is needed in order to assess the toxicity of residual magnetic nanopartilces in the grafts before Mag-TE can be used clinically.…”

“…We previously developed magnetite cationic liposomes (MCLs), which contain 10-nm magnetite nanoparticles, in order to improve accumulation of magnetite nanoparticles in target cells, due to electrostatic interaction between MCLs and the cell membrane (Shinkai et al, 1996). In addition, we recently developed a novel tissue engineering technique using MCLs, based on the fact that cells labeled with MCLs can be manipulated using magnetic force (Ito et al, 2004a(Ito et al, , 2004c(Ito et al, , 2005a(Ito et al, , 2005cShimizu et al, 2005). With this technique, we used a magnet to accumulate magnetically labeled keratinocytes onto a culture surface, and sheet-like multi-layered 3D constructs were successfully created without using any artificial polymer scaffolds (Ito et al, 2004a).…”

“…In addition, we recently developed a novel tissue engineering technique using MCLs, based on the fact that cells labeled with MCLs can be manipulated using magnetic force (Ito et al, 2004a(Ito et al, , 2004c(Ito et al, , 2005a(Ito et al, , 2005cShimizu et al, 2005). With this technique, we used a magnet to accumulate magnetically labeled keratinocytes onto a culture surface, and sheet-like multi-layered 3D constructs were successfully created without using any artificial polymer scaffolds (Ito et al, 2004a). Thus, we developed a novel methodology for tissue engineering using magnetite nanoparticles and magnetic force, which we designated "magnetic force-based tissue engineering (Mag-TE)".…”

Electronic Control of Cell Detachment Using a Self‐Doped Conducting Polymer

“…enzymatic treatments, these techniques provide temporal or spatial control for cell detachment [7,10] in cell sheet engineering [1,8,11,12] and cell co-culturing. [9,14] In addition to provide spatiotemporal control and to be less harmful to cells, an alternative detachment method should combine control over the detachment rate, cells fully released from the carrier substrate, no harsh conditions during actual detachment, and simple low-cost manufacturing.…”

Coating Nanomagnetic Particles for Biomedical Applications

“…Ito el al. proposed the use of magnetite cationic lipossomes (MCLs) in a novel methodology (Mag-TE) to construct multilayered keratinocyte sheets and collect the sheets without enzymatic treatment in presence of magnetic field. It was possible obtain keratinocytes further stratified and, subsequently, 10-layered epidermal sheets (Ito et al, 2004a). The technique Mag-TE was applied to construct heterotypic layered coculture system of rat hepatocytes and human aortic endothelial cells (HAECs).…”