High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures.

Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there is an urgent need for biologically relevant, in vitro high-throughput screening assays (HTS) to predict potential risks for drug-induced seizure early in drug discovery. We investigated drug-induced changes in neural Ca2+ oscillations, using fluorescent dyes as a potential indicator of seizure risk, in hiPSC-derived neurons co-cultured with human primary astrocytes in both 2D and 3D forms. The dynamics of synchronized neuronal calcium oscillations were measured with an FDSS kinetics reader. Drug responses in synchronized Ca2+ oscillations were recorded in both 2D and 3D hiPSC-derived neuron/primary astrocyte co-cultures using positive controls (4-aminopyridine and kainic acid) and negative control (acetaminophen). Subsequently, blinded tests were carried out for 25 drugs with known clinical seizure incidence. Positive predictive value (accuracy) based on significant changes in the peak number of Ca2+ oscillations among 25 reference drugs was 91% in 2D vs. 45% in 3D hiPSC-neuron/primary astrocyte co-cultures. These data suggest that drugs that alter neuronal activity and may have potential risk for seizures can be identified with high accuracy using an HTS approach using the measurements of Ca2+ oscillations in hiPSC-derived neurons co-cultured with primary astrocytes in 2D.

Noninvasive optical coherence tomography imaging of three-dimensional cardiac tissues derived from human induced pluripotent stem cells.

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.

Evaluation of the effects of cell-dispensing using an inkjet-based bioprinter on cell integrity by RNA-seq analysis.

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.

Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA.

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.

High-precision three-dimensional inkjet technology for live cell bioprinting.

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.

Development of vascularized iPSC derived 3D-cardiomyocyte tissues by filtration Layer-by-Layer technique and their application for pharmaceutical assays.

In vitro development of three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has long been desired in tissue regeneration and pharmaceutical assays. In particular, in vitro construction of 3D-iPSC-CM tissues with blood capillary networks have attracted much attention because blood capillaries are crucial for nutrient and oxygen supplies for CMs. Blood capillaries in 3D-iPSC-CM tissues will also be important for in vitro toxicity assay of prodrugs because of the signaling interaction between cardiomyocytes and endothelial cells. Here, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique for cells, instead of our previous centrifugation-LbL technique. The filtration-LbL allowed us to fabricate nanometer-sized extracellular matrices (ECM), fibronectin and gelatin (FN-G), films onto iPSC-CM surfaces without any damage and with high yield, although centrifugation-LbL induced physical stress and a lower yield. The fabricated FN-G nanofilms interacted with integrin molecules on the cell membrane to construct 3D-tissues. We found that the introduction of normal human cardiac fibroblasts (NHCFs) into the iPSC-CM tissues modulated organization and synchronous beating depending on NHCF ratios. Moreover, co-culture with normal human cardiac microvascular endothelial cells (NHCMECs) successfully provided blood capillary-like networks in 3D-iPSC-CM tissues, depending on NHCF ratios. The vascularized 3D-iPSC-CM tissues indicated significantly different toxicity responses as compared to 2D-iPSC-CM cells by addition of doxorubicin as a model of a toxic drug. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. STATEMENT OF SIGNIFICANCE: 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.