Consistent cell preparation is a fundamental preliminary step for understanding complex cellular mechanisms in various cell-based research fields, including basic cell biology, cancer research, and tissue engineering. However, certain elusive factors, such as cellular de-differentiation and contamination with mycoplasma or other types of cells, have compromised the reproducibility and reliability of cell-based approaches. Here, we propose an epi relief-contrast cellular monitoring system (eRC-CMS) that allows images of cells in a typical culture plate to be acquired, stored, and analysed for daily cell quality control. Due to its full flatbed nature and automated system, cells placed at any location on the stage can be analysed without special attention. Using this system, changes in the size, circularity, and proliferation of endothelial cells in subculture were recorded. Analyses of images of ~9,930,000 individual cells revealed that the growth activity and cell circularity in subcultures were closely correlated with their angiogenic activity in a subsequent hydrogel assay, demonstrating that eRC-CMS is useful for assessing cell quality in advance. We further demonstrated that eRC-CMS was feasible for the imaging of neurite elongation and spheroid formation. This system may provide a robust and versatile approach for daily cell preparation to facilitate reliable and reproducible cell-based studies.

Perfusable vasculatures are essential for engineering three-dimensional thick tissues and organs in the fields of tissue engineering and regenerative medicine. Here, we describe an approach for the fabrication of double-layered vascular-like structures (DVSs) composed of a monolayer of human vascular endothelial cells (HUVECs) covered with a dense human smooth muscle cell (SMC) layer. HUVECs were attached to a gold needle via the oligopeptide self-assembled monolayer and grown to form a HUVEC monolayer that was subsequently embedded in a photo-crosslinkable gelatin hydrogel containing SMC spheroids in a culture chamber. During four days of culture, the hydrogel significantly contracted and formed a dense SMC layer around the needle. The binding between the HUVEC layer and the gold needle was cleaved by applying a negative potential to desorb the oligopeptide and the needle was extracted from the chamber, resulting in a perfusable DVS composed of HUVEC and SMC layers. The DVS was cultured under perfusion, and the cells in the DVS showed greater expressions of SMC-specific genes compared to those of spheroids. The DVS possessed a dynamic contraction ability in response to acetylcholine as observed in the in vivo SMC layer. This study proposes a promising approach for the fabrication of perfusable vasculatures for the engineering of fully vascularized tissues and organs.

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