Modeling of human HSV infection: development of immune-competent 3D skin-on-chip with vascular perfusion

摘要 传统的机制研究和药物开发严重依赖于体内动物模型和体外 细胞培养系统。尽管传统的体外培养的2D或3D细胞能够分析信号 与感染或各种条件的反应相关的信号基因的途径和鉴定 和治疗，它们不能复制发生在组织中的复杂的细胞-细胞和细胞-基质相互作用 微环境。动物模型提供了对体内整合的多器官反应的理解，但 对它们的预测价值和与人类的生物学相关性存在严重关切。事实上，越来越多的 候选药物未能从第一阶段临床试验进展到第三阶段临床试验，并未能进入市场。批判性 减少临床试验中代价高昂的失败的挑战突显了为临床试验生成更好的模型系统的紧迫性 人类药物有效性和安全性的临床前测试，以及了解分子机制 导致了数千种人类疾病。一种三维(3D)人体组织模型有望令人信服 在预测复杂的生理功能、对传染病的免疫反应以及 对治疗剂的药理学反应。这种合成组织模型具有突出的潜力 可靠的药物疗效测试，作为动物模型的卓越替代品，特别是对那些感染的 没有足够的动物模型的疾病。皮肤是人体最大的器官，形成 保护身体免受病原体和潜在有害物质渗透的屏障。为了 模仿人类器官水平的皮肤病理生理学，我们建议利用生物工程方法来 开发一种体外3D“芯片上皮肤”，将循环免疫细胞整合到正常的 皮肤表皮和真皮用于模拟单纯疱疹病毒(HSV)中病毒与宿主的相互作用 感染。我们的具体目标是：1)建立和验证一个基于血管的3D芯片上皮肤平台 供体来源的原代细胞用于单纯疱疹病毒感染的建模和识别关键的免疫反应以进行保护。 2)将组织驻留的记忆T细胞室概括为3D血管化的芯片上的皮肤，用于建模 组织驻留记忆T细胞介导的局部免疫中的抗原特异性、细胞密度和TCR多样性 保护。

SUMMARY Conventional mechanistic research and drug development relies heavily on in vivo animal models and in vitro cell culture systems. Although conventional in vitro cultured 2D or 3D cells enable the analysis of signaling pathways and the identification of signature genes associated with responses of infection or various conditions and treatments, they can't reproduce complex cell-cell and cell-matrix interactions occurring in the tissue microenvironment. Animal models provide understanding on in vivo integrated multi-organ responses but serious concerns exist over their predictive value and biological relevance to humans. In fact, more and more drug candidates have failed to advance from Phase I to Phase III clinical trials and to reach the market. Critical challenges to reduce costly failures in clinical trials highlight the urgency to generate better model systems for preclinical testing of drug efficacy and safety in humans, and for understanding molecular mechanisms underlying thousands of human diseases. A three-dimensional (3D) human tissue model promises compelling advantages to predict complex physiological functions, immune responses to infectious diseases, and pharmacological responses to therapeutic agents. Such a synthetic tissue model has outstanding potential for reliable drug efficacy testing and as a superior replacement for an animal model, especially for those infectious diseases that do not have adequate animal models. Skin is the largest organ of the human body and forms a barrier to protect the body against pathogens and penetration of potential harmful substances. In order to mimic the organ-level skin pathophysiology in humans, we propose to harness bioengineering approaches to develop an in vitro 3D `skin-on-chip' that incorporates circulating immune cells into the normal architecture of skin epidermis and dermis for modeling of viral-host interactions in human herpes simplex virus (HSV) infection. Our Specific Aims are: 1) Establish and validate a vascularized 3D `skin-on-chip' platform using donor-derived primary cells for modeling of HSV infection and identifying key immune responses for protection. 2) Recapitulate a tissue resident memory T-cell compartment in 3D vascularized `skin-on-chip' for modeling of antigenic specificity, cell density and TCR diversity in tissue resident memory T-cell mediated local immune protection.