Collaborative Research: EAGER: 3D Bioprinted Organoids for Studying the Mechanism of Cerebrovascular Aging

认知健康与终生维持功能良好的脑血管系统有关。然而，脑血管系统中不同细胞的形态/结构和功能的年龄依赖性变化有助于不同衰老相关疾病的发展。衰老过程中的这些变化之一是诱导称为细胞衰老的复杂细胞应激反应。衰老细胞是经历代谢活性改变的活细胞，并且可以损害组织修复和再生过程，从而有助于衰老。将新型、高效且可重复的3D生物打印类器官用于脑血管模型将能够生产具有精确控制比例和不同类型衰老细胞的特定类器官。这种方法可以通过减少时间负担来模拟自然衰老或不同的神经退行性疾病，而无需将研究转移到冗长，更复杂和昂贵的体内实验中。最后，该项目将为各级学生提供多学科的良好教育培训机会，特别是通过让STEM中代表性不足的少数参与者参与并开展有趣和有效的外展活动。本研究的目标是开发由人类诱导多能干细胞（iPSC）衍生的神经元，星形胶质细胞，和微血管内皮细胞，以研究衰老细胞对衰老相关miRNA的差异表达和外泌体分泌的影响。细胞衰老被认为是组织退化的主要机制之一，也是许多年龄相关疾病的原因。使用基于微流体的3D细胞培养模型的创新方法将通过产生类似于年轻器官、老化前表型或老年类器官的3D类器官来研究衰老过程，这将通过在3D类器官生物打印过程中控制输入不同百分比的衰老细胞来实现。此外，将研究衰老细胞过表达衰老相关miRNA的类器官模型，用于miRNA模拟物的细胞递送，以调节健康类器官中衰老相关分泌表型（SASP）miRNA的表达。关于miRNAs在细胞衰老过程中的调控机制以及miRNAs在脑血管对衰老诱导物（如辐射暴露）的反应中的作用的数据很少。此外，这些机制和相关途径的体外研究一直非常具有挑战性，并且大多数研究仅限于二维（2D）培养系统，无法捕获复杂的血管组织功能。重要的是，成功完成这项高风险的研究衰老过程的新研究，将能够建立高度精确的模型，允许操纵和研究不同的与年龄有关的疾病，也将为发展与各种器官衰老有关的类似研究奠定基础，该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值进行评估来支持和更广泛的影响审查标准。

Cognitive health is associated with the maintenance of a well-functioning cerebrovascular system throughout life. However, age-dependent changes in morphology/structure and function in different cells in the cerebrovascular system contribute to the development of different aging-associated diseases. One of these changes during aging is the induction of a complex cellular stress response termed cellular senescence. Senescent cells are viable cells that undergo alterations in metabolic activity and can compromise tissue repair and regeneration processes, thereby contributing toward aging. The use of novel, highly efficient, and reproducible 3D bioprinted organoids for cerebrovascular models will enable production of specific organoids with precisely controlled proportions and different types of senescent cells. This approach can mimic natural aging or different neurodegenerative diseases by decreasing the time burden without moving the research onto lengthy, more complex, and expensive in vivo experiments. Finally, the project will provide excellent educational training opportunities in multi-disciplinary subjects for students at all levels, especially by involving underrepresented minority participants in STEM and the development of interesting and effective outreach activities.The goal of this research is to develop 3D cerebrovascular organoids composed of human-induced pluripotent stem cells (iPSC) derived neurons, astrocytes, and cerebromicrovascular endothelial cells to study the impact of senescent cells on differential expression and exosomal secretion of senescence-associated miRNAs. Cellular senescence is recognized as one of the major mechanisms of tissue deterioration and the cause of many age-associated diseases. The innovative approach of using a microfluidic-based 3D cell culture model will enable the study of the aging process by producing a 3D organoid resembling a young organ, a pre-aging phenotype, or an old organoid, which will be achieved by controlled input of different percentages of senescent cells during the 3D organoid bioprinting process. In addition, an organoid model with senescent cells overexpressing senescence-associated miRNAs will be studied for cellular delivery of miRNA mimics to regulate the expression of senescence-associated secretory phenotype (SASPs) miRNA in healthy organoids. There is little data on the regulatory mechanisms of miRNAs during the process of cellular senescence and the role of miRNAs in cerebrovascular responses to senescence inducers such as radiation exposure. Moreover, in vitro investigation of such mechanisms and associated pathways has been extraordinarily challenging, and most studies have been limited to two-dimensional (2D) culture systems failing to capture complex vascular tissue functionalities. Importantly, the successful completion of this high-risk novel research to study the aging process will enable the establishment of highly precise models allowing manipulations and studies of different age-related diseases and will also provide a base for developing similar studies related to senescence in various organ-specific studies.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.