Microfluidic tumor tissue processing platform for single cell diagnostics

摘要 实体瘤是不同细胞类型的多样性生态系统，这种异质性已被认为是肿瘤的一个重要特征。 疾病进展、转移和耐药性的关键因素。越来越多的单细胞分析 方法被用来定义肿瘤内的细胞亚群，以解决生物和治疗问题。 问题.然而，首先将组织转化为单细胞的需要是更广泛应用的显著障碍。 尤其是在临床环境中。目前的肿瘤解离方法耗时长，效率低， 规范此外，对于某些细胞亚型是否更容易释放， 这会使结果产生偏差。在以前的工作中，我们开发了新颖的微流体装置， 水动力将组织分解成单细胞。我们已经展示了出色的表现 使用体外肿瘤细胞聚集体和小鼠器官，显著增强单细胞恢复， 减少处理时间。在这个提议中，我们将开发一个集成的微流体平台， 从根本上改变肿瘤组织解离成单细胞的方式，从而促进单细胞诊断。 这将涉及四个独立的微流体设备技术，我们已经率先在出版或 前期工作这些设备被设计成按顺序工作，每个设备以不同的尺寸工作 规模从肿瘤组织标本（消化）开始，通过大的聚集体（解离）和集群 （过滤），最后洗脱100%单细胞的悬浮液（声洗脱）。任何剩余的细胞群 再循环回到装置的前端以最大化细胞回收。单个细胞将持续 解离后几分钟内，一旦准备好，就从系统中洗脱，以防止过度治疗 保持活力。我们将首先使用人类乳房分别开发和优化每种设备， 胰腺和前列腺肿瘤组织标本。接下来，我们将把所有设备集成到一个多功能系统中， 将操作一个、多个或所有设备，以及建立连续处理。最后，我们将严格 使用单细胞RNA测序（scRNAseq）评估悬浮液，以评估细胞亚型是否 在连续处理下，由具有不同时间进程的任何设备组件和/或微处理器偏置。的 本项目为期3年，具体目标包括：（1）优化使用人类肿瘤组织的微流控装置 （2）开发声洗脱装置，（3）整合所有装置并建立连续的 处理，和（4）使用scRNAseq评估装置处理的细胞的偏置和洗脱动力学。我们 微流控装置平台技术将直接影响肿瘤组织的单细胞分析， 新兴的和潜在的变革性方法scRNAseq。scRNAseq进入临床环境 将通过提供对细胞的初步调查， 预后和治疗特征的前景。我们的设备将通过自动化 解离工作流程，提高效率，最大限度地减少组织预处理，消除偏倚， 连续洗脱单个细胞。

ABSTRACT Solid tumors are diverse ecosystems of different cell types, and this heterogeneity has been implicated as a key factor driving disease progression, metastasis, and drug resistance. Increasingly, single cell analysis methods are being used to define cellular subsets within tumors to address biological and therapeutic questions. However, the need to first convert tissue into single cells is a significant barrier to more widespread use, particularly in clinical settings. Current tumor dissociation methods are long, inefficient, and not standardized. Moreover, there remains a question as to whether certain cell subtypes are easier to release than others, which would bias results. In previous work, we developed novel microfluidic devices that utilized hydrodynamic forces to break down tissue into single cells. We have already shown excellent performance using in vitro tumor cell aggregates and mouse organs, significantly enhancing single cell recovery and decreasing processed time. In this proposal, we will develop an integrated microfluidic platform that will radically change the way tumor tissue is dissociated into single cells, and thus facilitate single cell diagnostics. This will involve four separate microfluidic device technologies that we have pioneered in published or preliminary work. These devices were designed to work sequentially, with each operating at a different size scale starting from tumor tissue specimen (Digestion), through large aggregates (Dissociation) and clusters (Filter), and finally eluting a suspension of 100% single cells (Acousto-Elution). Any remaining cell clusters will be recirculated back into the front end of the device to maximize cell recovery. Single cells will be continuously eluted from the system as soon as they are ready, within minutes after dissociation, to prevent over treatment and maintain viability. We will first develop and optimize each device separately using human breast, pancreatic, and prostate tumor tissue specimens. Next we will integrate all devices into a versatile system that will operate one, multiple, or all devices, as well as establish continuous processing. Finally, we will rigorously evaluate suspensions using single cell RNA sequencing (scRNAseq) to assess whether cell sub-types are biased by any device component and/or elute with different time-courses under continuous processing. The Specific Aims for this 3 year project include: (1) optimize microfluidic devices using human tumor tissue specimens, (2) develop the Acousto-Elution Device, (3) integrate all devices and establish continuous processing, and (4) evaluate device processed cells for biasing and elution dynamics using scRNAseq. Our microfluidic device platform technology will directly impact single cell analysis of tumor tissues, including the emerging and potentially transformative method scRNAseq. Penetration of scRNAseq into clinical settings would help usher in an era of precision molecular medicine by providing an initial survey of the cellular landscape for prognostic and therapeutic signatures. Our device will advance these goals by automating the dissociation workflow, increasing efficiency, minimizing tissue pre-processing, eliminating bias, and continuously eluting single cells.