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: 10173403
• 负责人: Jered Brackston Haun
• 金额: $ 37.4万
• 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/10173403
• 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样化生态系统，这种异质性被认为是 驱动疾病进展、转移和耐药性的关键因素。单细胞分析越来越多 方法被用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细胞，这是更广泛应用的一个重大障碍。 使用，特别是在临床环境中。目前的肿瘤解离方法耗时长、效率低且不适合 标准化。此外，仍然存在一个问题，即某些细胞亚型是否更容易释放 比其他人更重要，这会导致结果出现偏差。在之前的工作中，我们开发了新颖的微流体装置，该装置利用 将组织分解成单个细胞的水动力。我们已经展现了出色的表现 使用体外肿瘤细胞聚集体和小鼠器官，显着增强单细胞恢复和 减少处理时间。在本提案中，我们将开发一个集成微流控平台，该平台将 从根本上改变肿瘤组织解离为单细胞的方式，从而促进单细胞诊断。 这将涉及我们在已发表或发表的文章中率先推出的四种独立的微流体设备技术。 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 从肿瘤组织样本（消化）开始，通过大聚集体（解离）和簇 (过滤)，最后洗脱100%单细胞悬浮液(Acousto-Elution)。任何剩余的细胞簇将 被再循环回到设备的前端，以最大限度地提高细胞回收率。单细胞将不断 一旦准备好，在解离后几分钟内就从系统中洗脱，以防止过度治疗 并保持活力。我们首先会利用人体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。接下来我们将把所有设备集成到一个多功能系统中 将操作一台、多台或所有设备，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，以评估细胞亚型是否 受到任何设备组件的偏差和/或在连续处理下以不同的时间过程进行洗脱。这 这个为期 3 年的项目的具体目标包括：（1）利用人体肿瘤组织优化微流体装置 样本，(2) 开发声洗脱装置，(3) 集成所有装置并建立连续的 (4) 使用 scRNAseq 评估设备处理细胞的偏置和洗脱动力学。我们的 微流控装置平台技术将直接影响肿瘤组织的单细胞分析，包括 新兴且具有潜在变革性的方法 scRNAseq。 scRNAseq 渗透到临床环境 通过提供对细胞的初步调查，将有助于开创精准分子医学时代 预后和治疗特征的景观。我们的设备将通过自动化来推进这些目标 解离工作流程，提高效率，最大限度地减少组织预处理，消除偏差，以及 连续洗脱单细胞。