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.

摘要