Several in vitro cell-based methods for predicting the skin sensitizing potential have been reported; however, such methods are limited by the poor water solubility of many organic molecules. Therefore, we developed a novel test method using a collagen vitrigel membrane (CVM) chamber, termed the Vitrigel Skin Sensitization Test (Vitrigel-SST). We first determined the optimal concentration of dimethyl sulfoxide (DMSO) for test chemical solutions subjected to the Vitrigel-SST. When medium containing 10% DMSO was subjected to the Vitrigel-SST using THP-1 cells, cytotoxicity was not observed until the 3 h time point. Thus, we decided to use 10% DMSO for Vitrigel-SST. Test chemicals were dispersed or dissolved in medium containing 10% DMSO. THP-1 cells were exposed to the test solutions for 30 min through the CVM, and the test solutions were replaced to fresh medium. After 48 h incubation, interleukin 8 (IL-8) productions was measured. Twenty-four test chemicals were evaluated to demonstrate the capacity of the Vitrigel-SST to predict chemical-induced skin sensitization. Cells exposed fifteen chemicals, including five chemicals with poor water solubility, increased maximum IL-8 production (IL-8MAX) more than 2-fold (≥2) that of cells exposed to the control treatment. IL-8MAX of five non-skin sensitizers was less than 2. The results of 20 of 24 chemicals were matched with that of LLNA when the IL-8MAX of 2 was adopted as cut off value. These results demonstrate that the Vitrigel-SST is a promising new skin sensitization test and is suitable to evaluate the skin sensitization potential of chemicals with poor water solubility.
We prepared multiple cell types from surplus finger tissues obtained from surgeries for polydactyly performed in Japanese patients. We tested their suitability as new bioresources for the National Institute of Biomedical Innovation, Health and Nutrition/Japanese Collection of Research Bioresources tissue bank. Dermal fibroblasts, epidermal keratinocytes, melanocytes, preadipocytes, and bone-derived cells were isolated from fresh or frozen tissues and subjected to quality control tests that assessed viability after frozen storage, morphology, microbial contamination, and cell-specific function. We confirmed that the cells exhibited the expected qualities and represented suitable research resources derived from Japanese individuals. Our public human tissue bank will provide these cells to academics and industries as useful alternatives to animal experiments in the research and development of drugs and cosmetics.
Human anatomy, physiology and biochemistry are categorized as one of the fundamental subjects for basic medical science, and students aiming for medical or paramedical field must learn them thoroughly at a relatively early phase in training school. The anatomy is especially a severe subject for many students because of its difficulties to image three-dimensional (3D) structure of the human body. Anatomical dissection of laboratory animals such as mouse or rat is one of the powerful approach to learn the anatomical 3D structure and must be an essential, irreplaceable experience for students. In order to observe more detail, fixation techniques are often used. In general, formaldehyde (formalin) is widely employed to fix animal specimens. However, because of the toxicity of formalin for human body or environment, it is not suitable for student's experiment and/or practice. In this report, we used more safety and less harmful animal fixation method than above mentioned traditional one by exchanging of fixation liquid to "Farmer's fixative". This liquid consists of ethanol and acetic acid (ethanol:acetic acid; 3:1, v:v). Although, this liquid usually used in fixation for plants or cultured cells, we applied in the whole animal body and evaluated for usage of student's anatomy practice in rat. In this report, we evaluated the quality or appearance of animals or tissues fixed by this method and showed that it can be fixed sufficiently necessary for detailed observation or dissection of various organ systems of rat. Thus, we propose that this method can be applied enough for the anatomy practice of rats for students aiming for medical or paramedical field. Furthermore, since this method can be used repeatedly for dissection training without scarifying new experimental living animals, it is possible to realize "Reduction" in 3Rs.