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

正在进行的新冠肺炎大流行突显了可用于研究该病毒的人类细胞培养模型的缺乏，以及这一缺陷对人类健康和疾病的破坏性后果。这项拟议的项目名为“新出现的呼吸道病原体和呼吸系统疾病活组织培养”，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等尖端技术，以了解新冠肺炎在肺部的传播。这将增加对SARS-CoV-2生物学和传播的知识。研究人员开发了这项技术，将部分肺组织培养成3D打印的活组织结构，类似于肺中发现的液体-固体基质中的结构。因此，与标准细胞培养相比，这个系统更接近于活人的环境。在这些样本感染SARS-CoV-2病毒后，这些“活组织切片”的高级成像将被用于研究病毒在细胞之间的传播以及治疗方法的有效性。拟议研究的成果包括：(I)使用人类肺部活检和已知的诊断方法验证标准模型系统，以应对SARS-CoV-OC43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。预计该系统将让科学家更好地了解病毒的传播和预防。该项目还支持对三名研究生的培训，从而增加未来推动生物经济的工作人员。提出团队假设，在人呼吸道微组织外植体3D培养模型中控制灌流SARS-CoV-2可以概括SARS-CoV-2感染和新冠肺炎病的早期阶段。为了验证这一假设，PI将使用活的人支气管和外周肺微组织外植体建立病毒感染的3D模型，使用新的3D组织培养平台量化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致病机制。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、病毒学和肺部生物学组成的跨学科研究团队。拟议的工作由两项任务组成。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、细胞因子产生和应对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-OC43、HCoV-NL63、SARS-CoV-2)和一种蝙蝠株(btCoV-HKU3)的人肺活检的时空致病机制。与细胞系的测量相比，活检的异质性将改变活检的病毒滴度和细胞因子的产生，并将提供比标准细胞培养技术更好的关于进展和病毒在组织中传播的信息。这一快速奖项是由综合组织系统生物部的生理和结构系统集群利用冠状病毒援助、救济和经济安全(CARE)法案的资金颁发的。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.