In the cosmetics industry, where the development and utilization of alternatives to animal testing is being promoted, a method to develop individual tests and evaluate them in combination according to the Key Events of the Adverse Outcome Pathway has been proposed and utilized as an alternative method for local toxicity. On the other hand, for systemic toxicity, recently, the concept of Next Generation Risk Assessment has been proposed, and there have been many reports on case studies that combine New Approach Methodologies tests in accordance with scenarios tailored to the characteristics of compounds, with the ultimate goal of protecting human health. Although many issues remain for alternative methods to animal testing, we would like to introduce some cases here, as they are expected to be developed and utilized in a variety of fields in the future.

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MPS（Microphysiological Systems） are emerging as highly promising systems in terms of the ability to mimic physiological conditions through perfusion culture via microfluidic devices and/or three-dimensional arrangement of human cells. In 2022, the FDA Modernization Act 2.0 marked a significant turning point in drug development, as it eliminated the mandatory use of animal testing and explicitly recognized MPS as an example of alternative testing methods. This shift indicates a paradigm change from traditional animal models toward innovative technologies that mimic human physiological functions. Going forward, particularly for modalities involving highly specific antibodies and cells that are significantly influenced by the microenvironment of target sites, it will be essential to appropriately select and apply both animal testing and its alternatives. This paper discusses the current status and future perspectives of MPS research and development in academia, focusing on the concepts of “alternative methods” and “human physiological mimetic systems.”

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In the drug discovery and development process, the use of experimental animals is crucial for evaluating pharmacokinetics, safety, and efficacy of drug candidates. There has been a growing demand for alternative methods from the perspective of animal welfare. Therefore, to enhance the extrapolation of preclinical research to humans, there is an increasing need for new in vitro models that can replicate human physiological responses. One of the promising technologies is the microphysiological systems（MPS）, which is expected to improve the success rates of drug development. This review outlines the impact of MPS on drug discovery and development and the needs for its utilization from the perspective of the pharmaceutical industry. Furthermore, examples of the application of MPS in drug delivery system research targeting the central nervous system will be introduced.

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In drug discovery research in the oncology field, in vitro models, ex vivo platforms and tumor-on-a-chip models using patient-derived tissues that provide highly predictive data for clinical settings have been developed. These advancements enable the elucidation of mechanisms of action and patient stratification, leading to rapid evaluation of new drug candidates and efficient translational research. Additionally, the use of high-content imaging technology and biobanks having various cancer tissues offers significant value to drug discovery research. By integrating these technologies into a comprehensive platform, it contributes to the 3Rs（refinement, replacement, and reduction of animal use） and simultaneously improves the success rates in clinical trials, reduces costs, and then enhances the efficiency of the pharmaceutical development process, resulting in accelerating the approval process of innovative new drugs.

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To address the limited clinical relevance of traditional animal models, we propose the use of advanced mouse models as refined in vivo platforms to improve translational outcomes. In this article, we highlight the utility of “humanized mice” including gene-humanized mouse models and immune-humanized NSG mice, which recapitulate human microenvironments and disease phenotypes in vivo. For those currently using or considering the use of mice, we also provide practical guidance on implementing the 3Rs in animal welfare（Replacement, Reduction, and Refinement） through appropriate colony management and strain selection. Although the complete elimination of animal experiments in preclinical studies remains a challenge, we hope this article helps you maximize the value of animal experiments while minimizing animal use to shape a better future.

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The TK6 human lymphoblastoid cell line, characterized by a wild-type p53 status, is widely recognized as a robust in vitromodel for genotoxicity testing. It is routinely employed in assays compliant with OECD test guidelines, specifically the in vitromicronucleus assay（TG 487） and the gene mutation assay（TG 490）. The presence of functional p53 contributes to the cell line’s reduced false-positive rate compared to rodent-derived cells, enhancing its relevance for human risk assessment. This biological fidelity makes TK6 cells particularly valuable in regulatory toxicology. Recent technological advances have further expanded the utility of TK6 cells. For example, integration with error-corrected next-generation sequencing（ecNGS） enables ultra-sensitive detection of mutational events at low frequencies, thereby improving the mechanistic understanding of genotoxic agents. Additionally, the MultiFlow assay—an advanced flow cytometry-based multiplex assay—facilitates the simultaneous detection of multiple genotoxicity endpoints, increasing throughput and data richness. These innovations have enabled more detailed mechanistic analyses and the identification of novel biomarkers for DNA damage and repair processes. Moreover, genome editing technologies have produced TK6-derived mutants deficient in specific DNA repair pathways, thereby broadening the application of this model to fundamental research on DNA damage responses and cellular repair mechanisms. Such modified cell lines provide powerful tools to dissect genotoxic mechanisms and to evaluate cellular effects of chemical compounds in a controlled genetic background. The reliability and versatility of TK6 cells have led to their growing acceptance in regulatory submissions across multiple jurisdictions. As animal welfare concerns intensify and regulatory frameworks increasingly endorse the 3Rs principles（Replacement, Reduction, Refinement）, TK6 cells represent a scientifically validated alternative to in vivogenotoxicity testing. This review comprehensively summarizes the current state of TK6 cell applications in genotoxicity assessment, highlights recent technological advancements, and discusses future perspectives for their expanded use as an alternative to animal experiments.

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In response to the increasing need for alternative methods to animal testing, the Japanese Center for the Validation of Alternative Methods（JaCVAM） was established in 2005 at the National Institute of Health Sciences in order to appropriately evaluate and verify them from the perspective of administrative utilization. In this paper, I first explain the role of JaCVAM, and in particular, show the actual validation process, which is essential for standardization, and the current status of the modernization to deal with complex toxicity such as systemic toxicity in the future. Furthermore, I will concretely introduce the process from AOP development to the development of a defined approach, using an OECD test guideline actually developed in Japan as an example. Finally, I will introduce the author’s efforts in the Japanese Pharmacopoeia and the Quasi-Drug Guidance as examples of standardization other than toxicity evaluation of chemical substances.

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JACEMIN is an autologous cultured epidermis maintaining melanocytes, approved for manufacture and sale in March 2023 and covered by insurance in October 2024 as a regenerative medical product. This product is produced by isolating and culturing epidermal cells, including melanocytes, from a patient’s own healthy skin tissue, forming a sheet structure. JACEMIN is transplanted into areas affected by vitiligo where nonsurgical therapy is ineffective or not indicated, after the epidermal layer has been removed（shaved thinly）. Through this transplantation, the patient’s own melanocytes are supplied along with epidermal cells, leading to repigmentation.

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