Microfluidic tumor tissue processing platform for single cell diagnostics

用于单细胞诊断的微流控肿瘤组织处理平台

• 批准号: 10398180
• 负责人: Jered Brackston Haun
• 金额: $ 36.6万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10398180
• 关键词: Acoustics; Address; Automobile Driving; Back; Biological Response Modifier Therapy; Buffers; Cell Extracts; Cell Survival; Cell physiology; Cells; Clinical; Devices; Diagnostic; Diagnostic Procedure; Digestion; Disease Progression; Dissection; Dissociation; Drug resistance; Ecosystem; Ensure; Flow Cytometry; Future; Goals; Heterogeneity; Human; In Vitro; Individual; Lasers; Mammary Neoplasms; Methods; Microfluidic Microchips; Microfluidics; Molecular Medicine; Mus; Neoplasm Metastasis; Organ; Patients; Penetration; Performance; Process; Production; Property; Prostatic Neoplasms; Publishing; Radiation; Recovery; Resistance; Role; Solid Neoplasm; Specimen; Stream; Surveys; Suspensions; System; Technology; Testing; Therapeutic; Time; Tissues; Tumor Biology; Tumor Tissue; Work; base; biological specimen archives; cell type; clinical diagnostics; design; drug sensitivity; hydrodynamic flow; innovation; interest; laser capture microdissection; microfluidic technology; neoplastic cell; novel; novel diagnostics; operation; overtreatment; pancreatic neoplasm; prevent; prognostic; prospective; public health relevance; single cell analysis; single cell sequencing; single-cell RNA sequencing; targeted treatment; tissue processing; transcriptome; tumor; tumor heterogeneity

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.

抽象的 实体瘤是不同细胞类型的多种生态系统，这种异质性已被视为 驱动疾病进展，转移和耐药性的关键因素。越来越多的单细胞分析 方法用于定义肿瘤中的细胞子集来解决生物学和治疗 问题。但是，首先将组织转换为单个细胞的需求是更广泛的显着障碍 使用，特别是在临床环境中。当前的肿瘤解离方法长，效率低下，而不是 标准化。此外，关于某些单元格是否更容易释放的问题仍然存在一个问题 比其他人，这会偏向结果。在以前的工作中，我们开发了使用的新型微流体设备 流体动力将组织分解成单细胞。我们已经表现出色 使用体外肿瘤细胞聚集体和小鼠器官，显着增强了单细胞恢复和 减少处理时间。在此建议中，我们将开发一个集成的微流体平台 从根本上改变了肿瘤组织分离为单个细胞的方式，从而促进了单细胞诊断。 这将涉及四种我们在发布或发表的单独的微流体设备技术 初步工作。这些设备旨在依次工作，每个设备的尺寸都不同 从肿瘤组织标本（消化）开始，从大骨料（解离）和簇开始 （滤波器），最后洗脱100％单细胞的悬浮液（声明）。任何剩余的细胞簇都将 循环回到设备的前端，以最大化单元格恢复。单细胞将连续 在解离后几分钟内准备就绪后从系统中洗脱，以防止过度治疗 并保持生存能力。我们将首先使用人乳房分别开发和优化每个设备， 胰腺和前列腺肿瘤组织标本。接下来，我们将将所有设备集成到多功能系统中 将操作一个，多个或所有设备，并建立连续处理。最后，我们将严格 使用单细胞RNA测序（SCRNASEQ）评估悬浮液，以评估细胞子类型是否为 在连续处理下，任何设备组件和/或洗脱的任何设备组件和/或洗脱。这 该3年项目的具体目的包括：（1）使用人肿瘤组织优化微流体设备 标本，（2）开发声明设备，（3）整合所有设备并建立连续的设备 处理，（4）使用SCRNASEQ评估设备处理的单元格，以进行偏置和洗脱动力学。我们的 微流体设备平台技术将直接影响肿瘤组织的单细胞分析，包括 新兴和潜在的变换方法scrnaseq。将scrnaseq穿透到临床环境中 通过对细胞的初步调查，将帮助迎接精确分子医学的时代 预后和治疗特征的景观。我们的设备将通过自动化这些目标来推进这些目标 解离工作流程，提高效率，最大程度地减少组织预处理，消除偏差，并 连续洗脱单细胞。