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

摘要 老年性黄斑变性(AMD)是导致老年人严重视力障碍的主要原因 50发达国家[1，2]。干细胞来源的视网膜色素上皮(RPE)移植 目前治疗视网膜变性和晚期非新生血管性AMD(NNAMD)的一种有希望的方法[3- 6]。已经开发了许多从人类多能干细胞中获得RPE的方案 胚胎干细胞(HESC)或人类诱导多能干细胞(IPSC)[7-12]。产品的质量控制 在临床试验中，供体细胞是细胞生产的基本要求。干细胞残留物与染色体 长期培养过程中的数量变化必须在临床使用前进行测试。然而，茎的质量控制 细胞残留物(多能性)和干细胞致瘤性不是微不足道的。物理移除RPE中的单元 培养过程中的单层会由于上皮-间充质的转变而导致单层的萎缩。 (EMT)和伤口愈合。在空间RPE植入物的分子图谱方面有一个未得到满足的需求 RNA测序(RNA-seq)。聚焦超声(FUS)为这种未得到满足的需求提供了一种解决方案，因为它可以 通过含有细胞的液滴从固体表面喷射细胞，对细胞的影响最小 弹出点周围的边缘。超声波在液体和固体中传播，FUS换能器 不一定要与细胞生长的底物物理接触。的单元格数量 FUS换能器的射出取决于FUS的焦点大小，它可以非常小，并且非常 精确度高，重复性好。此外，它是低成本和有效的分离数十到数百个单细胞在 通过一组换能器并行工作。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学换能器(SFAT)来实现无损伤、含细胞 用于空间单细胞RNA序列的生长在薄固体支架上的RPE单层的液滴喷射。此外 对于RPE植入物生产的质量控制，SFAT前所未有的按需排出 微粒子或细胞(直径几十到几百微米)将打开许多新的可能性 空间分子细胞研究、基因转染、并列和操纵。

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.