The surface of biomaterials with a fine micro/nano-pattern reportedly affects cell behavior. Chitosan was previously shown to exhibit antibacterial properties and thus here we evaluated the cellular response and antibacterial activity of chitosan micro/nano patterns prepared by the imprint method. We fabricated micro/nano patterns with different sizes and shapes of pillars, grooves, and holes. The chitosan pattern films exhibited antibacterial activity towards Streptococcus mutans. In addition, we observed mammalian cells adhering to and spreading on the chitosan pattern, and found that the orientation of the cells was controlled by the shape of the pattern.

We previously succeeded in fabricating a microvascular endothelial model with excellent endothelial barrier function by coculturing human microvascular endothelial cells (HMVECs) and human dermal fibroblasts (HDFs) via a collagen vitrigel membrane (CVM). The disadvantage of this model is the low reproducibility especially due to the low proliferation potential of HMVECs. Meanwhile, HMEC-1 cells, a well-known immortalized HMVEC line, show low endothelial barrier function although they have high proliferating potential. This study aimed to create a hybrid cell line by fusing HMVECs and HMEC-1 cells. Hybrid cells were composed of relatively uniform cobblestone-shaped cells compared to HMEC-1 cells and proliferated well. Hybrid cells cultured in a CVM chamber for 14 days highly expressed vascular endothelial (VE)-cadherin at the plasma membrane while VE-cadherin was mainly expressed in the cytoplasm in HMEC-1 cells. The transendothelial electrical resistance (TEER) value of the coculture model of hybrid cells and HDFs via a CVM gradually increased and reached over 30 Ω･cm² on day 14. On the other hand, the value of the coculture model of HMEC-1 cells and HDFs via a CVM reached a plateau at approximately 10 Ω･cm². In conclusion, hybrid human microvascular endothelial cells prepared by a cell fusion technique can express high endothelial barrier function and may be useful for quantitative analyses of microvascular permeability.

As an evaluation of the biological safety of nanomaterials, in vitro experimental methods basically do not have the complicated problems of animal welfare compared to animal experiments, and may be statistically accurate. Predictive effects on human embryotoxicity are one of the most difficult areas in the biological safety assessment of nanomaterials. There is a possibility that models for the prediction of embryotoxicity can be produced by in vitro tests. In 1997, Spielmann et al. proposed the first in vitro embryotoxicity test method using mouse ES-D3 and 3T3 cells. We have proposed various methods that are further improvements of the EST method. We investigated the utilization of feeder cells, which was not available in the EST protocol for application to other type ES cells.

The biocompatibility of nanomaterials is quite different from that of body fluid-soluble chemicals. Nanomaterials have a large astronomical surface area relative to volume, and the mechanism by phagocytosis also affects them. For special toxicity such as embryotoxicity of nanomaterials, continuous research results are desired in the future.