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

摘要 视网膜相关性黄斑变性（AMD）是导致老年人严重视力损害的主要原因 50个发达国家[1，2]。干细胞衍生的视网膜色素上皮（RPE）的移植是 目前是治疗视网膜变性和晚期非新生血管性AMD（NAMD）的有前景的方法[3- 4]。 6]。已经开发了许多方案用于从来自人的多能干细胞衍生RPE。 胚胎干细胞（hESC）或人诱导多能干细胞（iPSC）[7-12]。的质量控制 供体细胞是临床试验中细胞生产的基本要求。干细胞残留物和染色体 临床使用前必须检测长期培养过程中的数量变化。然而，股骨柄的质量控制 细胞残基（多能性）和干细胞致瘤性并非微不足道。从RPE中物理移除细胞 由于上皮-间充质转化，在培养期间单层将导致单层的营养不良 (EMT)和伤口愈合。在RPE植入物的分子谱分析中存在未满足的需求，其具有空间结构。 RNA测序（RNA-seq）。聚焦超声（FUS）为这一未满足的需求提供了解决方案，因为它可以 通过含有细胞的液滴从固体表面喷射细胞， 围绕喷射点的边缘。超声波通过液体和固体传播，FUS换能器 不必与细胞生长的基底物理接触。细胞的数量 由FUS换能器发射的能量取决于FUS的焦点尺寸，其可以非常小，并且非常小。 精确且可重复。此外，它是低成本的，并且有效地用于分离数十至数百个单细胞， 平行通过换能器阵列。满足未满足的需求，实现RPE细胞治疗 对于AMD，我们建议使用自聚焦声换能器（SFAT）进行无损伤、含细胞的 用于空间单细胞RNA-seq的从在薄固体支架上生长的RPE单层的液滴喷射。除了 对于RPE植入物生产的质量控制，SFAT前所未有的按需喷射能力， 微粒或细胞（直径为数十至数百微米）将为人类的免疫学研究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。

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.