Conventional methods of skin stimulus evaluation for cosmetics are performed by animal tests. Alternatives to animal tests are proposed to achieve animal welfare and for example, reconstructed human epidermis (RHE) models are approved officially. However, they are based on endpoint assay using a dye causing large test errors. Furthermore, no single method is proposed for all tests of skin corrosion, irritation and sensitization. Epidermal keratinocyte communicates with other cells using mitochondria exotically emitting both intracellular and extracellular danger signals depending on the stimulus. Here, we monitored mitochondrial membrane potential (MMP) change in keratinocyte by a cosmetic compound addition real-timely without labeling using our original in vivo-like technology of high–precision surface plasmon resonance (HP-SPR)-three dimension (3D). We compared MMP signal trend to the conventional methods results and examined the feasibility of HP-SPR-3D for the universal method against various skin stimuli. Using five cosmetic compounds, the MMP trends until 10 min and after 30 min of the compound addition expressed the acute mode of action and In vivo score values including sensitization, respectively. These were further confirmed by the cell morphological observation after the test. Many compounds are expected to be measured using the HP-SPR-3D system for the cosmetics universal validation.
Hepatic stellate cells play a cardinal role in the development of liver fibrosis. Quiescent hepatic stellate cells isolated from normal liver are activated by plating on a plastic culture dish. Therefore, a culture method that maintains hepatic stellate cells in a quiescent state is required for studies of fibrosis. We attempted to deactivate human hepatic stellate cells by culturing on VECELL® culture inserts (Preset VECELL). VECELL is a cell culture scaffold consisting of an expanded polytetrafluoroethylene mesh that is coated with collagen type I. Cryopreserved human hepatic stellate cells and LI90 cells, which is a cell line established from an outgrowth of a human hepatic mesenchymal tumor, were cultured on Preset VECELL. The expression of activation markers α-SMA and COL1A was decreased by VECELL cultivation. In addition, actin filaments (markers of activated hepatic stellate cells) were not detected in LI90 cells on VECELL inserts. These results suggest that human hepatic stellate cells were deactivated by VECELL cultivation, which could provide a model system for the analysis of deactivated human hepatic stellate cells. Thus, Preset VECELL will be a useful in vitro tool for the clarification of underlying mechanisms and the development of drugs to treat liver fibrosis. This study will contribute to provide alternative methods to animal tests that have been mainly carried out in studies of hepatic stellate cell, liver fibrosis, and liver cirrhosis.