3D Culture Systems Of Urine-Derived Stem Cell For NTRI-Induced Mitotoxicity Assessment

用于 NTRI 诱导的细胞毒性评估的尿源干细胞 3D 培养系统

• 批准号: 10083026
• 负责人: YUANYUAN no ZHANG
• 金额: $ 19.38万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083026
• 关键词: 3-Dimensional; Accounting; Affect; Animal Model; Animals; Anti-HIV Agents; Anti-Retroviral Agents; Antiviral Agents; Attention; Biological Assay; Biological Sciences; Cell Culture System; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Survival; Cells; Chronic; Clinic; Clinical; Clinical Trials; Collaborations; Detection; Development; Disease; Dose; Drug Evaluation; Drug Screening; Drug toxicity; Environment; Evaluation; Exhibits; Genetic Transcription; Goals; Growth; HIV; HepG2; Hepatocyte; Human; In Vitro; Individual; Injury; Institutes; Integrase; Life; Life Expectancy; Longevity; Measures; Metabolism; Mitochondria; Mitochondrial DNA; Modeling; Monitor; Morbidity - disease rate; Organoids; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Ploidies; Preclinical Drug Development; Preclinical Drug Evaluation; Primary Cell Cultures; Property; Regimen; Respiration; Safety; System; Telomerase; Tenofovir; Testing; Therapeutic; Tissue Banks; Toxic effect; Toxicity Tests; Toxicology; Urine; Zidovudine; antiretroviral therapy; base; cell growth; clinical predictors; clinically relevant; cost; cost effective; cytotoxicity; drug development; drug testing; early phase clinical trial; human stem cells; human tissue; improved; in vitro Assay; in vivo; insight; monocyte; mortality; outcome prediction; peripheral blood; personalized medicine; pre-clinical; preclinical study; preclinical toxicity; procedure cost; progenitor; screening; side effect; stem; stem cells; stemness; three dimensional cell culture; tool; two-dimensional

Summary Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic life expectancy for individuals with HIV increased, the long-term safety of ART has gained increasing attention. Thus, it is highly important to the long-term safety of antiretroviral regimens. Human hepatocyte cell lines in 2D culture are most used to evaluate short-term mitochondrial toxicity (MtT) induced by ART. However, these cannot be used in detection of ART-induced chronic MtT because they cannot survive more than days during toxicity testing. In addition, antiviral drug toxicity screening often requires expensive primary such as peripheral blood monocytes in 2D culture systems. More than 90% of drugs that pass through in 2D culture preclinical studies fail to meet the desired efficacy or safety margins required in subsequent trials. Clearly, 2D cultures have contributed to the poor predictive power of MtT screening assays in . Although long-term toxicity tests are often performed in animal models, the high rates of MtT observed in earlier clinical trials suggest that animal toxicology studies may not be suitable for predicting MtT of antiviral intended for human use. Apparently, there are no adequate 2D cell culture or animal models for late MtT for preclinical drug development. Hence, there is an urgent need to develop more toxicologically and clinically predictive in vitro assays to assess compounds for the potential of late MtT. We were the to demonstrate that stem/progenitor cells exist in human urine, i.e., urine-derived stem cells (USC). These can be obtained using simple, non-invasive and low-cost procedures. USC express telomerase activity possess robust proliferative potential. We recently revealed that 3D culture of USC provides a long-term, microenvironmen for cell growth and proliferation, mimicking in vivo conditions, which may have to improve the predictive outcome of preclinical antiviral drug studies. Thus, is to develop 3D culture systems of human USC for ART-induced MtT testing. We hypothesize that human USC maintain telomerase activity and mitochondrial function in 3D organoids, which considerably extends the life of cell culture systems, enabling long-term assessment of late ART-induced MtT . To test this hypothesis, we propose the following: Aim 1. Assess the stemness properties and mitochondrial function of human USC in 3D organoids over long-term culture, compared to 2D culture of USC; Aim 2. Determine the cytotoxicity, inhibition of Pol-γ, mitochondrial DNA content and MtT profiles of 9 antiretroviral drugs in 3D USC cultures. We that the antiretroviral drugs tested will exhibit effects on MtT in the 3D culture systems and be ranked to their effective concentrations based on in vitro MtT assay. Therefore, the use of patients-derived cells from urine to generate 3D culture assay offers a promising tool for antiretroviral drug development personalized medicine in the assessment of MtT of anti-HIV drugs. benefit of ART is often limited by delayed drug-associated toxicity. As has monitor commonly hepatocytes 14 cells vitro clinical vitro the drugs evaluation relevant first cells and better t potential the goal of this project anticipate according stem and

概括 抗逆转录病毒治疗（ART）显着降低了艾滋病毒相关的发病率和死亡率。然而，治疗 艾滋病毒感染者的预期寿命 增加，ART 的长期安全性越来越受到关注。因此，非常重要的是 抗逆转录病毒疗法的长期安全性。二维培养的人肝细胞系是大多数 用于评估 ART 诱导的短期线粒体毒性 (MtT)。然而，这些 不能用于检测 ART 诱导的慢性 MtT，因为它们的存活时间不能超过 毒性测试期间的天数。此外，抗病毒药物毒性筛查通常需要昂贵的初级检查 例如二维培养系统中的外周血单核细胞。 90%以上的药物通过 2D 培养临床前研究未能满足后续所需的疗效或安全裕度 试验。显然，2D 培养导致 MtT 筛选试验的预测能力较差。 。尽管长期毒性试验经常在动物模型中进行，但在动物模型中观察到的高 MtT 发生率 早期的临床试验表明动物毒理学研究可能不适合预测抗病毒药物的 MtT 供人类使用。显然，没有足够的 2D 细胞培养物或动物模型来进行晚期 MtT 用于临床前药物开发。因此，迫切需要开发更多的毒理学 以及临床预测性体外测定，以评估化合物晚期 MtT 的潜力。我们是 证明人尿液中存在干/祖细胞，即尿源干细胞（USC）。这些 可以通过简单、非侵入性和低成本的程序获得。 USC 表达端粒酶活性 具有强大的增殖潜力。我们最近透露，南加州大学的 3D 文化提供了长期的、 细胞生长和增殖的微环境，模仿体内条件，这可能有 提高临床前抗病毒药物研究的预测结果。因此， 旨在开发人类 USC 3D 培养系统，用于 ART 诱导的 MtT 测试。我们假设人类 USC 在 3D 类器官中维持端粒酶活性和线粒体功能，这大大扩展了 细胞培养系统的寿命，从而能够对晚期 ART 诱导的 MtT 进行长期评估。为了检验这个假设， 我们提出以下建议： 目标 1. 评估人类 USC 的干性特性和线粒体功能 长期培养的 3D 类器官与南加州大学 2D 培养相比；目标 2. 确定细胞毒性， 3D USC 培养物中 9 种抗逆转录病毒药物的 Pol-γ 抑制、线粒体 DNA 含量和 MtT 谱。我们 测试的抗逆转录病毒药物将在 3D 培养系统中表现出对 MtT 的影响并进行排名 根据体外 MtT 测定得出其有效浓度。因此，使用源自患者的 从尿液中提取细胞生成 3D 培养测定为抗逆转录病毒药物开发提供了一种有前景的工具 个性化医疗在抗 HIV 药物 MtT 评估中的应用 ART 的益处常常受到延迟药物相关毒性的限制。作为 有 监视器 通常 肝细胞 14 细胞 体外 临床 体外 这 药物 评估 相关的 第一的 细胞 和 更好 该项目的潜在目标 预料 根据 干 和