RAPID: CLEARED: Culture of Living-biopsies for Emerging Airway-pathogens and REspiratory Disease

持续的COVID-19大流行凸显了缺乏可用于研究这种病毒的人类细胞培养模型，以及这种缺陷对人类健康和疾病的破坏性后果。拟议的项目被称为“CLEARED”，用于新兴气道病原体和呼吸道疾病的活体活检培养，结合了3D打印，软组织工程，人工智能增强成像，人类肺部生物学和病毒学的尖端技术，以了解COVID-19在肺部的传播。这将增加SARS-CoV-2生物学和传播的知识。研究人员已经开发出一种技术，将肺的一部分生长成活的3D打印组织结构，这种结构类似于肺中的液体状固体基质。因此，与标准细胞培养物相比，该系统更接近于活体人类的环境。在用SARS-CoV-2病毒感染这些样本后，这些“活组织检查”的先进成像将用于研究病毒在细胞间的传播以及治疗性治疗的功效。拟议研究的结果包括：（i）使用人类肺活检和已知诊断方法验证标准模型系统，以应对SARS-CoV-OC 43感染;（ii）确定疾病如何在感染不同人类和蝙蝠冠状病毒株的活检中发展和传播。预计该系统将使科学家更好地了解病毒传播和预防。该项目还支持培训三名研究生，从而增加未来的工作人员，以推动生物经济。提议团队假设，在人类呼吸道微组织外植体的3D培养模型中控制SARS-CoV-2的灌注可以重现SARS-CoV-2感染和COVID-19疾病的早期阶段。为了验证这一假设，PI将使用人类支气管和外周肺的活微组织外植体建立病毒感染的3D模型，使用新型3D组织培养平台量化对病毒感染的早期反应，并确定不同人类和蝙蝠冠状病毒株的时空发病机制。初步数据显示，SARS-CoV-2确实感染了支气管和外周肺的微组织。这是一个由天文学、化学、医学、工程学、病毒学和肺生物学的研究人员组成的跨学科团队。 拟议的工作分为两项任务。任务1将使用人类肺活检和已知的宿主对SARS-CoV-2感染的反应来验证标准模型系统。读数将包括病毒滴度、细胞因子产生和响应冠状病毒感染的病毒复制的时空成像。任务2将确定感染不同人类冠状病毒株（HCoV-OC 43、HCoV-NL 63、SARS-CoV-2）和一种蝙蝠株（btCoV-HKU 3）的人类肺活检标本的时空发病机制。与细胞系中的测量相比，活检的异质性将改变活检的病毒滴度和细胞因子产生，并且将提供比标准细胞培养技术上级的关于进展和病毒通过组织传播的信息。该RAPID奖项由BIO综合有机系统部门的生理和结构系统集群颁发，使用冠状病毒援助，救济和经济安全（CARES）法案的资金。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The ongoing COVID-19 pandemic has highlighted the lack of human cell culture models available for studying this virus, and the devastating consequences of this shortcoming as it relates to human health and disease. The proposed project, known as “CLEARED” for Culture of Living-biopsies for Emerging Airway-pathogens and REspiratory Disease, combines cutting-edge technologies in 3D-printing, soft tissue engineering, artificial-intelligence-enhanced imaging, human lung biology, and virology to understand the spread of COVID-19 in lungs. This will increase the knowledge of SARS-CoV-2 biology and transmission. The researchers have developed the technology to grow portions of lung into living 3D-printed tissue structures that resembles the architecture found in the lung in a liquid-like-solid matrix. Thus, this system more closely resemble the environment in living humans versus standard cell culture. After infecting these samples with SARS-CoV-2 virus, advanced imaging of these “living biopsies” will be used to study virus spread from cell to cell, and the efficacy of therapeutic treatments. Outcomes of the proposed research include: (i) Validating a standard model system using human lung biopsies and known diagnostics in response to SARS-CoV-OC43 infection; (ii) Determining how the disease develops and spreads in biopsies infected with different human and bat coronavirus strains. It is expected that this system will allow scientists to better understand virus transmission and prevention. This project also supports the training of three graduate students, leading to an increase in future workers to drive the bioeconomy.The proposing team hypothesizes that controlled perfusion of SARS-CoV-2 in 3D culture models of human respiratory microtissue explants can recapitulate early stages of SARS-CoV-2 infection and COVID-19 disease. To test this hypothesis, PIs will establish a 3D model of viral infection using living microtissue explants of human bronchus and peripheral lung, quantify the early responses to viral infection using a novel 3D tissue culture platform, and determine the spatiotemporal pathogenesis of different human and bat coronaviruses strains. Preliminary data show that SARS-CoV-2 indeed infects the micro-tissues of bronchus and peripheral lung. This is a transdisciplinary team of investigators from Astronomy, Chemistry, Medicine, Engineering, Virology and lung biology. The proposed work is organized by two tasks. Task 1 will validate a standard model system using human lung biopsies and known host-response to SARS-CoV-2 infection. Readouts will include viral titer, cytokine production and spatiotemporal imaging of viral replication in response to coronavirus infection. Task 2 will determine the spatiotemporal pathogenesis of human lung biopsies infected with different human coronavirus strains (HCoV-OC43, HCoV-NL63, SARS-CoV-2) and one bat strain (btCoV-HKU3). The heterogenous nature of biopsies will alter the viral titer and cytokine production of biopsies compared to measurements in cell lines, and will provide superior information about progression and virus spread through tissues than standard cell culture technology. This RAPID award is made by the Physiological and Structural Systems Cluster in the BIO Division of Integrative Organismal Systems, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.