Large Scale Nanochannel Electroporation (NEP) for Cell Reprogramming

用于细胞重编程的大规模纳米通道电穿孔 (NEP)

• 批准号: 8583897
• 负责人: Ly James Lee
• 金额: $ 23.03万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8583897
• 关键词: Accounting; Address; Adult; Biochemical; Biological; Biological Assay; Cell Count; Cell Lineage; Cell Survival; Cell Therapy; Cell model; Cells; Charge; Chemicals; Clinic; Complex; DNA; Development; Devices; Diffusion; Disease; Disease model; Electroporation; Experimental Models; Fibroblasts; Gene Expression; Generations; Goals; Hand; Harvest; Heterogeneity; Human; Interdisciplinary Study; Lead; Medical; Messenger RNA; Methods; Microinjections; Modeling; Motor Neurons; Nanotechnology; Nature; Neurons; Nuclear; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pluripotent Stem Cells; Population; Process; Property; RNA; Regenerative Medicine; Research; Stem cells; Stochastic Processes; Structure; System; Techniques; Technology; Testing; Time; Tissue Engineering; Transfection; Variant; Viral Vector; Visceral; base; cDNA Expression; cell injury; cell type; dosage; electric field; induced pluripotent stem cell; innovation; interest; meetings; nanochannel; nanoscale; nerve stem cell; new technology; novel; nuclear reprogramming; pluripotency; polycation; prevent; public health relevance; regenerative; reparative medicine; stem cell fate; tool; transdifferentiation; vector

DESCRIPTION (provided by applicant): Cell reprogramming holds great promise for a number of medical and biological applications, including regenerative/reparative medicine and cellular disease models. Significant progress has been made in this field since the introduction of induced pluripotent stem cells (iPSCs) and the subsequent development of directed nuclear reprogramming (i.e., transdifferentiation) approaches. However, nuclear reprogramming technologies have not been used to date to treat patients due to several obstacles, among them, the high heterogeneity and sometimes inherent unpredictability of the reprogrammed cell population, which is largely due in part to the inability to control the quantity and combination o the transfected reprogramming factors (DNA- or mRNA- based). A number of transfection methods, biological (e.g., viral vectors), chemical (e.g., lipoplexes, polycations) and physical (e.g., microinjection, electroporation), have been developed; however, the great majority of these techniques, with the exception of microinjection, are based on stochastic processes that lead to random cell transfection with significant cell-to-cell variations. Microinjection on the oter hand is only compatible with relatively large cells, and has low yields. New technologies capable of delivering reprogramming factors in a controlled (i.e., timing and dosage), safe, and efficient manner, at the single cell level, are clearly needed in this field for successful transition from te lab bench to the clinic. Our recently developed nanochannel-based electroporation (NEP) technology meets these criteria, thus potentially making it a powerful tool for this purpose. Here we propose to build upon our unique expertise on NEP to develop a more robust and versatile 3D system that could be implemented in a wide range of cell reprogramming applications. We will first implement modeling and micro/nanoscale technologies to develop an optimum 3D NEP platform that can support sequential transfection of large cell numbers (¿106), and then we will test this platform using induced pluripotency and direct neuronal transdifferentiation as nuclear reprogramming models. Finally, we will use our NEP technology to methodically study a number of aspects of the cell reprogramming process that cannot be addressed using conventional transfection technologies.

描述（由申请人提供）：细胞重编程在许多医学和生物学应用中具有很大的前景，包括再生/修复医学和细胞疾病模型。自从引入诱导多能干细胞（iPSC）和随后开发定向核重编程（即，转分化）方法。然而，由于若干障碍，核重编程技术迄今尚未用于治疗患者，其中包括重编程细胞群体的高度异质性和有时固有的不可预测性，这主要部分是由于无法控制转染的重编程因子（基于DNA或mRNA）的量和组合。许多转染方法，生物学的（例如，病毒载体），化学的（例如，脂质复合物，聚阳离子）和物理（例如，显微注射、电穿孔）;然而，除了显微注射之外，这些技术中的绝大多数是基于随机过程，其导致具有显著细胞间变化的随机细胞转染。另一方面，显微注射仅适用于相对较大的细胞，产量较低。能够在受控的（即，定时和剂量）、安全和有效的方式，在单细胞水平上，在该领域中显然需要从实验室工作台到临床的成功过渡。我们最近开发的基于纳米通道的电穿孔（NEP）技术符合这些标准，因此可能使其成为实现这一目的的强大工具。在这里，我们建议利用我们在NEP方面的独特专业知识，开发一个更强大和通用的3D系统，可以在广泛的细胞重编程应用中实现。我们将首先实施建模和微/纳米级技术，以开发一个最佳的3D NEP平台，该平台可以支持大细胞数量的顺序转染（约106），然后我们将使用诱导多能性和直接神经元转分化作为核重编程模型来测试这个平台。最后，我们将使用我们的NEP技术来系统地研究细胞重编程过程的一些方面，这些方面无法使用常规转染技术来解决。