Three‐dimensional (3D) printers are attracting attention as a method for arranging and building cells in three dimensions. Bioprinting technology has potential in tissue engineering for the fabrication of scaffolds, cells, and tissues. However, these various printing technologies have limitations with respect to print resolution and due to the characteristics of bioink such as viscosity. We report a method for constructing of 3D tissues with a “microscopic painting device using a painting needle method” that, when used with the layer‐by‐layer (LbL) cell coating technique, replaces conventional methods. This method is a technique of attaching the high viscosity bioink to the painting needle tip and arranging it on a substrate, and can construct 3D tissues without damage to cells. Cell viability is the same before and after painting. We used this biofabrication device to construct 3D cardiac tissue (LbL‐3D Heart) using human‐induced pluripotent stem cell–derived cardiomyocytes. The constructed LbL‐3D Heart chips had multiple layers with a thickness of 60 µm, a diameter of 1.1 mm, and showed synchronous beating (50–60 beats per min). The aforementioned device and method of 3D tissue construction can be applied to various kinds of tissue models and would be a useful tool for pharmaceutical applications.

In this study, three-dimensional (3D) cardiac tissue constructed using the pin type bioprinter ‘microscopic painting device’ and layer-by-layer cell coating technique was confirmed to have drug responsiveness by three different analytical methods for cardiotoxicity assay. Recently, increasing attention has been focused on biofabrication to create biomimetic 3D tissue. Although various tissues can be produced in vitro, there are many issues surrounding the stability and reproducibility of the preparation of 3D tissues. Thus, although many bioprinters have been developed, none can efficiently, reproducibly and precisely produce small 3D tissues (μm-mm order) such as spheroids, which are most commonly used in drug development. The 3D cardiac tissue chips were successfully constructed with a similar number of cells as conventional 2D tissue using a pin type bioprinter, and corresponding drug-induced cardiotoxicities were obtained with known compounds that induce cardiotoxicity. The 3D cardiac tissue chips displayed uniform cell density and completely synchronized electrophysiological properties as compared to 2D tissue. The 3D tissues constructed using a pin type bioprinter as a biofabrication device would be promising tools for cardiotoxicity assay as they are capable of obtaining stable and reproducible data, which cannot be obtained by 2D tissue.