Embedded Printing of Human Respiratory Model with Air-Liquid Interface for COVID-19 Research

项目摘要 这项研究的首要目标是构建一个体外三维(3D)的人类呼吸系统 气液界面(ALI)模型，可作为新冠肺炎相关生物医学的平台 调查。最近的新冠肺炎疫情，这是由于感染了一种严重的急性呼吸道疾病 综合症冠状病毒2(SARS-CoV-2)，导致1亿多确诊病例和约230万人 全球死亡人数每天都在增加。对于新冠肺炎的相关研究，仍然有一个紧迫的问题 需要一种3D完全异质的细胞呼吸模型，它可以增强我们对 SARS-CoV-2诱导肺损伤并促进作为动物补充的新治疗方法的开发 模型和临床患者。因此，我们建议开发这样的人类呼吸模型，其中1) 表达血管紧张素转换酶2蛋白和跨膜型丝氨酸蛋白酶2 (TMPRSS2)允许病毒进入以研究SARS-CoV-2感染过程，以及2)具有扩散 呼吸道和主要流体通道之间的渗透性，以使质量传输通过ALI，允许 新冠肺炎临床验证药物的筛选。我们假设一个体外的人类呼吸道 模型可以通过嵌入打印和进一步开发屈服应力中的可灌装结构来实现 含有原代人类基质细胞、内皮细胞和成纤维细胞的基质浴液。 因此，提出了两个具体目标如下： 目的1：基于基质细胞的3D体外人体呼吸模型的嵌入打印和灌流 可交叉连接的屈服-应力矩阵浴。 目的2：通过检测ACE2受体和TMPRSS2来表征人类呼吸模型 呼吸道上皮中的蛋白水解酶与ALI弥漫性通透性的测定。 首次提出了一种明胶微凝胶和明胶溶液为基础的可交联屈服应力细胞 将创新复合基质浴缸，以便在其中嵌入打印可灌流的组织结构，从而实现 人体呼吸系统模型的制作。这项模型开发研究将产生一个体外3D人体 利用新型可交叉连接屈服-应力细胞基质的ALI和细胞间质呼吸模型 BASH用于嵌入式打印，并且不存在具有这种生理复杂性的其他型号。这样的一个人 呼吸道模型将为研究新冠肺炎大流行相关感染提供通用的体外平台 相关药品的加工和筛选。

Project Summary The overarching goal of this research is to fabricate an in vitro three-dimensional (3D) human respiratory model with an air-liquid interface (ALI), which can serve as a platform for COVID-19 related biomedical investigations. A recent COVID-19 outbreak, which is due to the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), results in more than 100 million confirmed cases and about 2.3 million deaths with numbers increasing everyday globally. For COVID-19 related research, there remains an urgent need for a 3D fully heterogeneous, cellular respiratory model, which can enhance our understanding of how SARS-CoV-2 induces lung injury and facilitate the development of new treatments as complementary to animal models and clinical patients. We therefore propose to develop such a human respiratory model, which 1) expresses angiotensin-converting enzyme 2 (ACE2) protein and transmembrane protease serine type 2 (TMPRSS2) that allow viral entry to study the SARS-CoV-2 infection process, and 2) has the diffusional permeability between the airway and primary fluid channel to enable the mass transport across ALI, permitting the screening of clinically proven drugs for COVID-19 use. We hypothesize that an in vitro human respiratory model can be realized by embedded printing and further developing a perfusable construct in a yield-stress matrix bath containing primary human stromal, endothelial, and fibroblast cells. Accordingly, two specific aims are proposed as follows: Aim 1: Embedded printing and perfusion of a 3D in vitro human respiratory model in a stromal cell-based cross-linkable yield-stress matrix bath. Aim 2: Characterization of the human respiratory model via detection of ACE2 receptor and TMPRSS2 protease in the airway epithelium and determination of the diffusional permeability of ALI. For the first time, a gelatin microgels and gelatin solution-based cross-linkable yield-stress cellular composite matrix bath will be innovated for embedded printing of perfusable tissue constructs in it, enabling human respiratory model fabrication. This model development study will result in an in vitro 3D human respiratory model with ALI and cellular stroma by utilizing the novel cross-linkable yield-stress cellular matrix bath for embedded printing, and no other models with this physiological complexity exist. Such a human respiratory model will provide a versatile in vitro platform for studying the COVID-19 pandemic-related infection process and screening related drugs.