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 年龄相关性黄斑变性（AMD）是老年人严重视力障碍的主要原因 发达国家有50%采用干细胞来源的视网膜色素上皮（RPE）移植。 目前是治疗视网膜变性和晚期非新生血管性 AMD (NNAMD) 的一种有前景的方法 [3- 6] 已经开发了许多用于从人类多能干细胞中衍生 RPE 的方案。 胚胎干细胞（hESC）或人诱导多能干细胞（iPSC）的质量控制[7-12]。 供体细胞是临床试验中细胞生产的基本要求 干细胞残留物和染色体。 临床使用前必须测试长期培养过程中的数量变化，但要进行干细胞的质量控制。 细胞残留（多能性）和干细胞致瘤性从物理上去除 RPE 中的细胞并非易事。 培养过程中单层细胞会因上皮-间质转化而导致单层细胞营养不良 （EMT）和伤口愈合对 RPE 植入物的空间分子分析的需求尚未得到满足。 RNA 测序 (RNA-seq) 为这一未满足的需求提供了解决方案。 通过含有细胞的液滴从固体表面喷射细胞，对细胞的影响最小 喷射点周围的超声波通过液体和固体以及 FUS 传感器传播。 不必与细胞生长的基质发生物理接触。 FUS 换能器喷射的光量取决于 FUS 的焦点尺寸，该尺寸可能非常小，而且非常小。 此外，它可以低成本且有效地分离数十至数百个单细胞。 通过传感器阵列并行满足未满足的需求并实现 RPE 细胞疗法。 对于 AMD，我们建议使用自聚焦声换能器 (SFAT) 来实现无损伤、含有细胞 从生长在薄固体支架上的 RPE 单层喷射液滴，用于空间单细胞 RNA 测序。 为了控制 RPE 植入物生产的质量，SFAT 具有前所未有的按需弹出功能 微粒或细胞（直径数十至数百微米）将开辟许多新的可能性 空间分子细胞研究、基因转染、并置和操作。