Inkjet printing technology and bio-ink development for the biofabrication of in vitro 3D tissues

Bioprinting is an attractive technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. Particularly, piezoelectric drop-on-demand inkjet heads are expected to allow the arrangement of living cells with high precision. However, implementing a biofabrication process based on inkjet printing requires the consideration of several specific issues: first, nozzle clogging and loss of cell viability due to the sedimentation of cells inside the printing head; second, the need for selecting suitable bio-ink materials that would be compatible with droplet ejection at low viscosity and rapid tissue assembly. To address the first issue, we have developed a novel inkjet head specially designed for ejecting live cell suspensions. Droplets are formed by membrane vibrations generated by a pulse-driven piezoelectric actuator. Soft vibrations during the non-ejecting period also prevent cell sedimentation inside the chamber. Good stability of ejection has been thus achieved with over 90% viability when using human fibroblasts or endothelial cells. Next we have demonstrated the feasibility of constructing stratified mille-feuille like structures, composed of 2-10 cell layers with a total thickness of 50-300 um, by alternating cell suspension and hydrogel deposition. We are also investigating new bio-ink materials to improve tissue functionality without compromising reliability of ejection. For example, incorporation of submicron-sized gelatin beads into alginate gels has been shown to improve cell adhesion to printed substrates

A Novel Plasmid Carrying Capsule Gene Cluster Found in Lactococcus garvieae Isolated from Filefish

Lactococcus garvieae is recognized as a crucial bacterial pathogen of freshwater and marine fish species. It has been divided into two serological phenotypes, namely KG? and KG+. Difference of the two phenotypes is owing to the presence or absence of polysaccharide capsule, and a phenotypic change from KG? to KG+ occurs during stocking of isolates for a long period or by repeated subculturing. We found that the phenotypic change occurred more readily in L. garvieae isolates from cultured filefish, thread-sail filefish Stephanolepis cirrhifer and black scraper Thamnaconus modestus, than those from other fish species. Thus we studied the gene cluster for capsular polysaccharide biosynthesis (capsule gene cluster) of a filefish isolate, strain BSLG13015, and revealed that the strain possessed the same capsule gene cluster as those from other fish species, but that it was integrated in a newly identified plasmid. The plasmid, a size of 31,654 bp and circular, was named pBSLG13015. It was detected in all of KG? filefish isolates but not in KG+ filefish isolates or L. garvieae from other fish species. It is highly probable that the easier change from KG? to KG+ in L. garvieae filefish isolates is attributed to the loss of the plasmid