Damage-Free, Ultrasonic Cell Isolation from Retinal Pigment Epithelium (RPE) Monolayers

从视网膜色素上皮 (RPE) 单层中进行无损伤超声波细胞分离

• 批准号: 10717828
• 负责人: EUN SOK KIM
• 金额: $ 56.68万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10717828
• 关键词: Acoustics; Age; Age related macular degeneration; Area; Atrophic; Biopsy; Cell Separation; Cell Therapy; Cells; Chromosomes; Clinical; Clinical Trials; Collection; Complex; Data; Derivation procedure; Developed Countries; Development; Diameter; Encapsulated; Epithelial Cells; Fingerprint; Focused Ultrasound; Functional disorder; Gel; Genes; Goals; Graft Rejection; Human; Immune; Implant; Individual; Interruption; Liquid substance; Measures; Methods; Microfluidics; Microspheres; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Nature; Needles; Oils; Pathology; Patients; Persons; Photoreceptors; Pluripotent Stem Cells; Polymerase Chain Reaction; Population; Production; Progressive Disease; Protocols documentation; Quality Control; Radiation; Reagent; Research; Retina; Retinal Degeneration; Reverse Transcription; Shapes; Solid; Spottings; Stem cell transplant; Structure of retinal pigment epithelium; Surface; Suspensions; Technology; Temperature; Testing; Thinness; Transducers; Transfection; Transplantation; Tumorigenicity; Ultrasonic Transducer; Ultrasonics; Variant; Visual impairment; cell injury; cost; embryonic stem cell; epithelial to mesenchymal transition; human embryonic stem cell; improved; induced pluripotent stem cell; monolayer; novel; operation; particle; pluripotency; pressure; prevent; regenerative; scaffold; single-cell RNA sequencing; stem cells; subretinal injection; transcriptome; transcriptome sequencing; ultrasound; wound healing

Abstract Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in people over age 50 in developed countries [1,2]. Transplantation of stem cell derived retinal pigment epithelium (RPE) is currently a promising method to treat retinal degeneration and advanced non-neovascular AMD (NNAMD) [3- 6]. Many protocols have been developed for the derivation of RPE from pluripotent stem cells from human embryonic stem cells (hESC) or human induced pluripotent stem cell (iPSC) [7-12]. The quality control of donor cells is a basic requirement for cell production in clinical trials. Stem cell residues and chromosome number variation during long-term culture must be tested before clinical use. However, quality control for stem cell residues (pluripotency) and stem cell tumorigenicity is not trivial. Physically removing cells from an RPE monolayer during culture will result in hypotrophy of the monolayer due to epithelial-mesenchymal transition (EMT) and wound healing. There is an unmet need in the molecular profiling of RPE implants with spatial RNA sequencing (RNA-seq). A focused ultrasound (FUS) offers a solution to this unmet need, as it can produce ejection of cells via cell-containing liquid droplets from a solid surface with minimum impact on the edges surrounding the ejection spot. Ultrasound propagates through liquid and solid, and the FUS transducer does not have to be in physical contact with the substrate where cells are grown. The number of cells that are ejected by a FUS transducer depends on the focal size of the FUS, which can be very small, and is very precise and repeatable. Further, it is low-cost and effective for isolating tens to hundreds of single-cells in parallel through an array of transducers. To satisfy the unmet need and allow realization of RPE cell therapy for AMD, we propose to use self-focusing acoustic transducers (SFATs) for damage-free, cell-containing droplet ejection from RPE monolayers grown on thin solid scaffolds for spatial single-cell RNA-seq. Besides for quality control in RPE implant production, the SFAT’s unprecedented capability of on-demand ejection of microparticles or cells (of tens - hundreds of microns in diameter) will open up many new possibilities in spatial molecular cell study, gene transfection, juxtaposition and manipulation.

抽象的 与年龄相关的黄斑变性（AMD）是年龄段人们严重视力障碍的主要原因 发达国家有50个[1,2]。干细胞衍生的视网膜色素上皮（RPE）的移植IS 目前是治疗视网膜变性和晚期非血管AMD（NNAMD）的承诺方法[3- 6]。已经开发了许多方案，用于从人类的多能干细胞中衍生RPE 胚胎干细胞（HESC）或人类诱导的多能干细胞（IPSC）[7-12]。质量控制 供体细胞是临床试验中细胞生产的基本要求。干细胞保留和染色体 长期培养过程中的数量变化必须在临床使用前进行测试。但是，STEM的质量控制 细胞残基（多能性）和干细胞肿瘤性并非微不足道。从RPE物理去除细胞 培养过程中的单层将导致由于上皮 - 间质转变导致单层萎缩 （EMT）和伤口愈合。 RPE的分子分析有未满足的需要空间 RNA测序（RNA-Seq）。专注的超声（FUS）为这种未满足的需求提供了解决方案 通过含细胞的液滴从固体表面产生细胞的生产，对固体表面产生最小影响 弹出点周围的边缘。超声通过液体和固体传播，并发fus传感器 不必与细胞生长的底物进行物理接触。单元的数量 FUS换能器弹出的取决于FU的焦点，这可能很小，非常小 精确且可重复。此外，它是低成本且有效的 通过一系列换能器并行。满足未满足的需求并允许实现RPE细胞疗法 对于AMD，我们建议使用自我关注的声音传感器（SFAT）进行无损伤，细胞 从薄固体支架上生长的RPE单层的液滴射血，用于空间单细胞RNA-seq。除了 为了控制RPE植入物生产的质量控制 微粒或细胞（数十个 - 直径数百微米）将在许多新的可能性中开放 空间分子细胞研究，基因转染，并置和操纵。