NSF/FDA SIR: Assurance of Cellular Function in High-Shear Three-Dimensional Bioprinting

尽管药物和医疗器械在动物身上进行了严格的测试，但许多产品在早期的人体测试中失败了。这些失败的产品有可能导致试验参与者的不良健康后果，并导致每年数十亿美元的资本损失。制造人造人体组织样本是预测人类对新的、受监管的产品反应的一种潜在方式。这些制造工艺被设计为使用特殊的生物打印机以三维(3D)结构打印人类细胞。其目标是将这些合成组织作为另一个筛查步骤，在新医疗产品进入人体测试之前揭示其潜在危害。最终目标将是只有最安全、最有效的产品才能进行人体测试。然而，3D打印过程会对细胞施加机械力，导致细胞功能障碍或死亡。这个项目是肯塔基大学生物材料研究人员与美国食品和药物管理局的细胞生物力学和生物打印专家合作完成的，目的是确定3D打印程序如何改变细胞的行为方式。细胞生理和行为的变化反过来会影响人造组织在医疗产品筛选中的有效性。作为这项工作的结果，研究人员将能够更好地预测打印细胞是否会恢复其自然功能，以及打印后这种功能将在多长时间内恢复。有了这些新的信息，研究人员将能够开发和评估在组织制造过程中保存正常细胞功能的新方法。在这个项目中，研究小组还将开发公开可用的教育工具，描述三维生物打印背后的科学和工程概念。这个项目试图确定3D生物打印如何影响细胞生理学，特别是细胞膜运输。众所周知，生物打印喷嘴的高剪切环境会影响细胞反应；然而，细胞的确切变化以及在一系列生物打印参数范围内是否能恢复正常功能尚未得到评估。这种知识上的差距使得很难确定3D打印组织是否真的会复制正常(或定义的病理)生理--如果这种生物打印的组织结构将被用于药物或医疗器械的临床前评估，这是一个关键特征。这一提议的科学目标是：1)量化模拟挤压生物打印后跨细胞膜的非自然运输的大小和时间尺度，包括多种形式的主动和被动运输；以及2)量化假定用于保护细胞免受机械损伤的细胞涂层如何影响基于挤压的细胞膜运输的变化。除了膜转运的测量外，还将对每种细胞类型进行基本细胞功能和活性的适当分析。实验将使用五种细胞系(HepG2-肝脏；Caco-2-肠道；H9C2-心肌；A549-人肺癌；以及间充质干细胞)进行，每一种细胞都是目前生物制造领域的兴趣。这种合作将导致相互教育，博士后研究员在监管事务领域接受培训，PI在FDA培训CDRH(设备和放射健康中心)团队成员关于封装细胞系统和设备的新兴技术。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。

Despite rigorous testing of drugs and medical devices in animals, many products fail in early stage human testing. These failed products have the potential to result in adverse health outcomes for trial participants and billions in lost capital each year. The manufacturing of artificial human tissue samples is a potential way of predicting human response to new, regulated products. These manufacturing processes are being designed to use special bioprinters to print human cells in three dimensional (3D) constructs. The goal is to use these synthetic tissues as another screening step to reveal potential hazards of new medical products before they enter human testing. The eventual goal would be that only the safest, most effective products reach human testing. The 3D printing process, however, exerts mechanical forces on the cells and leads to cellular dysfunction or death. This project is a collaboration between researchers in biomaterials at the University of Kentucky and cellular biomechanics and bioprinting experts at the United States Food & Drug Administration to determine how 3D printing procedures may change the way cells behave. Changes in cellular physiology and behavior can, in turn, affect the usefulness of the artificial tissues for medical product screening. As a result of this work, researchers will be able to better predict if printed cells will return to their natural function and how long after being printed this function will be restored. With this new information, researchers will be able to develop and evaluate new methods to preserve normal cellular function during tissue manufacturing. During this project, the research team will also develop publicly available, educational tools that describe the science and engineering concepts underlying three dimensional bioprinting.This project seeks to determine how 3D bioprinting affects cellular physiology, in particular cellular membrane transport. It is known that the high shear environment of the bioprinting nozzle impacts cellular response; however, the precise changes in the cells and whether normal function can be recovered within a range of bioprinting parameters has not been assessed. This gap in knowledge makes it difficult to determine if a 3D printed tissue actually will replicate normal (or defined pathological) physiology -- a key feature if such bioprinted tissue structures are going to be used for pre-clinical assessment of drugs or medical devices. The scientific objectives of this proposal are to: 1) quantify the magnitude and timescale of unnatural transport across the cell membrane, including multiple forms of active and passive transport, following simulated extrusion bioprinting; and 2) quantify how cellular coatings, which are hypothesized to protect cells from mechanical damage, affect the extrusion-based alterations in cellular membrane transport. In addition to measurements of membrane transport, appropriate assays for basic cell function and viability will also be conducted for each cell type. Experiments will be conducted using five cell lines (HepG2 - hepatic; Caco2 - intestinal; H9C2 - myocardial; A549 - human lung carcinoma; and mesenchymal stem cells), each of which is of current interest in the biomanufacturing arena. This collaboration will result in mutual education, with the post-doctoral fellow becoming trained in the area of regulatory affairs and the PI educating CDRH (Center for Devices and Radiologic Health) team members at the FDA on emerging technology for encapsulated cell systems and devices.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.