Fabrication of 3D Cell Supporting Structures With Multi-Materials Using the Bio-Printer

Abstract

We produced three-dimensional (3-D) cell supporting structures for use in engineering regenerative living tissue. The structures were formed by alginate gel, a type of hydro-gel, and our originally developed printer, termed a 3-D bio-printer. A droplet ejected from an inkjet printer nozzle has the same size as several cells. Thus, we considered that such a printer nozzle would be able to eject cultured living cells, along with growth factor, protein, and other materials. If a 3-D gel structure could be formed with such cells, and the materials and cells self-assembled, a variety of living tissues could be obtained. In this report, our 3-D bio-printer and the method of fabrication of multiple material gel structures is presented. Our 3-D bio-printer has a printing mechanism that operates in 3 directions with a positioning resolution of 0.5 micrometers, which is adequate for precise positioning of the ejected cells. Further, a piezoelectric inkjet nozzle head that does not became heated during operation is used and cells can be ejected without heating. The head has 4 nozzles and is able to eject 4 different kinds of materials simultaneously. We used a sodium alginate solution and ejected it from the inkjet nozzle into a calcium chloride solution as a substrate, thus alginate gel beads were obtained. Several types of gel structures could be constructed by distributing the beads precisely and the resolution of the gel structures was as small as the size of an individual bead, about 10–60 μm in diameter. By continuous operation of the inkjet nozzle, gel lines were able to be formed. The substrate was a 10% calcium chloride solution on a slide glass and the ejected-droplets contained a 0.8% sodium alginate solution. In addition, gel sheets were formed by parallel gel lines. In that case, a 10% calcium chloride solution was also used as the substrate. It is possible that such 3-D gel sheet structures could be constructed by lamination in a high viscosity substrate. We also formed gel rings, which were stacked and allowed to sink into the substrate, thus obtaining gel tubes. By utilizing gel tubes with inner and outer walls formed using different types of gels with vascular endothelial cells and smooth muscle cells, blood vessel structures could be fabricated with the present system.