In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN-G) films onto iPSC-CM surfaces. The FN-G nanofilms induce cell-cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.

In recent years, bioprinting has emerged as a promising technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. In the present study, we present the development of an inkjet-based bioprinting system to arrange multiple cells and materials precisely into structurally organized constructs. A novel inkjet printhead has been specially designed for live cell ejection. Droplet formation is powered by piezoelectric membrane vibrations coupled with mixing movements to prevent cell sedimentation at the nozzle. Stable drop-on-demand dispensing and cell viability were validated over an adequately long time to allow the fabrication of 3D tissues. Reliable control of cell number and spatial positioning was demonstrated using two separate suspensions with different cell types printed sequentially. Finally, a process for constructing stratified Mille-Feuille-like 3D structures is proposed by alternately superimposing cell suspensions and hydrogel layers with a controlled vertical resolution. The results show that inkjet technology is effective for both two-dimensional patterning and 3D multilayering and has the potential to facilitate the achievement of live cell bioprinting with an unprecedented level of precision.

Bioprinting technology is expected to be applied in the fields of regenerative medicine and drug discovery. There are several types of bioprinters, especially inkjet-based bioprinter, which can be used not only as a printer for arranging cells but also as a precision cell-dispensing device with controlled cell numbers similar to a fluorescence activated cell sorter (FACS). Precise cell dispensers are expected to be useful in the fields of drug discovery and single-cell analysis. However, there are enduring concerns about the impacts of cell dispensers on cell integrity, particularly on sensitive cells, such as stem cells. In response to the concerns stated above, we developed a stress-free and media-direct-dispensing inkjet bioprinter. In the present study, in addition to conventional viability assessments, we evaluated the gene expression using RNA-seq to investigate whether the developed bioprinter influenced cell integrity in mouse embryonic stem cells. We evaluated the developed bioprinter based on three dispensing methods: manual operation using a micropipette, FACS and the developed inkjet bioprinter. According to the results, the developed inkjet bioprinter exhibited cell-friendly dispensing performance, which was similar to the manual dispensing operation, based not only on cell viability but also on gene expression levels.Bioprinting technology has grown in tandem with stem cell research, and it has become a vital tool with diverse applications in the biological and medical fields. Artificial tissues or organs fabricated using bioprinters are expected to be used in regenerative medicine and drug discovery 1,2 . There are several types of bioprinters, and inkjet-based bioprinters possess a specification to print a single cell 3 or cell aggregates 4 by controlling process parameters, such as nozzle diameter, droplet volume and cell concentration 5 . Owing to the specification mentioned above, inkjet-based bioprinter can be used not only as a printer for arranging cells but also as a precision cell-dispensing device with controlled cell numbers.Precise cell-dispensing technology is expected to be particularly useful in the fields of drug discovery and single-cell analysis. In the field of drug discovery, high-throughput screening of candidate drugs using cell microarrays 6 is expected to contribute to the reduction of costs for the development of new drugs. Cell microarrays containing some types of cells, especially disease-specific induced pluripotent stem cells (iPSCs) with controlled cell numbers on a chip, are expected to be applied to screen drug efficacy evaluation, toxicity testing and RNAi 7,8 In addition, cell arrays containing individually-derived iPSCs are expected to be used as personalised pharmaceutics tools 2 . In the field of single-cell analysis, technology for precise and high-throughput dispensing of single-cell is required with remarkable progress of single-cell genome sequencing and single-cell RNA-seq 9-11 . FACS is a mainstream tool in the field of precision cell dispensing with contr...

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as “cell number-based DNA reference material”). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

Artificial three-dimensional (3D) tissues have the potential to be used in regenerative medicine or in vitro screening. In particular, the fabrication of 3-D cardiac tissues is greatly anticipated. However, hierarchical organization of 3-D tissues is still unknown. In regenerative medicine and drug discovery, noninvasive evaluation methods of 3-D tissues including inside of it play a key role. In this study, we report on noninvasive methods of analyzing bio-fabricated 3-D cardiac tissues using optical coherence tomography (OCT) and image analysis. Three-dimensional cardiac tissues were fabricated by coating of extracellular matrix nanofilms onto a cell surface using a layer-by-layer (LbL) technique. At first, we investigated the relationship between surface beating and its thickness to assess the value of internal analysis. The results showed that the surface beating was influenced by the thickness. Next, we tried to quantitatively evaluate the internal beating of 3-D cardiac tissues. We also confirmed the methods by changing the beating properties through the administration of isoproterenol. Our results demonstrated that the beating properties of 3-D cardiac tissues differed by depth. The results of this study suggest that information on the internal properties of 3-D cardiac tissue was necessary to understand how it functions. The combination of OCT and image analysis can be used to evaluate the internal beating properties, including changes in beating induced by a drug. It is suggested that OCT and image analysis have the potential to be used as noninvasive methods in regenerative medicine and pharmaceutical applications.