Microfluidic tumor tissue processing platform for single cell diagnostics

摘要 实体瘤是不同细胞类型的不同生态系统，这种异质性被认为是 推动疾病进展、转移和抗药性的关键因素。越来越多的单细胞分析 正在使用方法来定义肿瘤内的细胞亚群，以解决生物学和治疗问题 问题。然而，首先需要将组织转化为单个细胞是更广泛应用的重大障碍 使用，特别是在临床环境中。目前的肿瘤分离方法冗长、低效，而且不是 标准化。此外，还有一个问题是，某些细胞亚型是否更容易释放 这会对结果产生偏差。在以前的工作中，我们开发了新型微流控设备，利用 流体动力将组织分解成单个细胞。我们已经表现出了出色的表现 利用体外肿瘤细胞聚集体和小鼠器官，显著提高单细胞回收率和 缩短处理时间。在这项提案中，我们将开发一个集成的微流控平台，它将 从根本上改变肿瘤组织分离成单细胞的方式，从而促进单细胞诊断。 这将涉及四种独立的微流控设备技术，我们已在发布或 前期工作。这些设备被设计为按顺序工作，每个设备以不同的大小运行 从肿瘤组织标本(消化)开始，到大的聚集体(解离)和簇 (过滤器)，最后洗脱100%单细胞悬浮液(声洗脱)。任何剩余的细胞集群都将 再循环到设备的前端，以最大限度地提高细胞回收率。单个细胞将连续 一旦它们准备好，在解离后的几分钟内从系统中洗脱，以防止过度治疗 并保持生存能力。我们将首先使用人的乳房分别开发和优化每个设备， 胰腺、前列腺癌组织标本。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，我们将严格执行 使用单细胞RNA测序(ScRNAseq)评估悬液以评估细胞亚型是否 被任何设备组件偏置和/或在连续处理下以不同的时间进程洗脱。这个 这个为期3年的项目的具体目标包括：(1)利用人类肿瘤组织优化微流控设备 (2)开发声洗脱装置；(3)集成各种装置，建立连续的 处理，以及(4)使用scRNAseq评估设备处理的细胞的偏置和洗脱动力学。我们的 微流控设备平台技术将直接影响肿瘤组织的单细胞分析，包括 新兴的和潜在的变革性方法scRNAseq。ScRNAseq在临床环境中的渗透 通过提供对细胞的初步调查，将有助于开创精确分子医学的时代 预见性和治疗性信号的景观。我们的设备将通过自动化 分离工作流程，提高效率，最大限度地减少组织前处理，消除偏见，以及 连续洗脱单细胞。

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.