TR&D-1: Cell Fabrication

项目概要/摘要： 概述：培养的细胞构成了几乎所有TEMPs和再生医学治疗的基础。 本TR&D的长期目标是开发新的技术和策略，以实现自动化的文化， 对于特定TE应用，表型和功能优化的细胞。选择单元格， 一致的属性，包括差异化和扩展潜力，是众所周知的困难，因为变量 供体遗传学和缺乏对细胞对其环境的反应的理解。 为了克服这些局限性，我们集中在（1）动态的，可诱导的分子报告和非侵入性传感器 以真实的时间跟踪和控制细胞和组织制造工作流程，以及（2）靶向修改 细胞内途径和细胞富集，导致更可预测和可控的细胞表型， 功能我们将从人类间充质干细胞开始开始;但是这些策略可以很容易地应用于，例如，到 组织特异性干细胞或iPS细胞。这将遵循三个具体目标： 目的1：产生用于动态和连续感测细胞生物学特性的遗传编码细胞传感器。 表型和功能。我们将产生荧光和/或生物发光（BLI）分子报告特异性 我们的CP基于miRNA和启动子驱动，这些记者会发出。 BLI可用于检测早期分化事件中的基因表达活性，作为追踪这些基因表达的可量化数据。 实时事件。高分辨率荧光成像允许生物标志物的时空评估 在单细胞水平上表达。TR&D-1将与TR& D-2、-3和-4合作，以验证他们的技术， 提供比通常使用的端点破坏性评估更高的吞吐量。 目的2：研制超灵敏、无创的细胞功能检测分子传感器。我们 将开发分子传感器和报告器，以在非常低的浓度下检测特定的生物分子 通过荧光和电化学传感系统，在细胞内或由细胞实时产生。我们 经验证的通用电化学生物传感器将适用于使用适体的任何感兴趣的分子 CRISPR-Cas 12（13）平台的高特异性。这些非侵入性测量将被设计用于 如TR&D-4中所述，集成到Tissue Foundry自动化主干中。 目的3：实现细胞表型和功能的动态调控。在评估了时空 - 使用实时分子报告物（SA-1和SA-2）的分化动力学，诱导型遗传控制和/或 将进行细胞的富集和预处理。这将通过使用诱导系统来实现 例如CRISPR激活、Tet-inducible系统和miRNA模拟/拮抗剂。交替细胞群 可以通过新的分选技术富集所需的生物学特征（例如，微流体 免疫磁性分选、无标记富集）。

Project Summary/Abstract: Overview: Cultured cells form the basis of almost all TEMPs and Regenerative Medicine-based therapeutics. The long-term goal of this TR&D is to develop new technologies and strategies to achieve automated culture of cells that are phenotypically and functionally optimized for a particular TE application. Selecting cells with consistent attributes, including differentiation and expansion potential, is notoriously difficult because of variable donor genetics and a lack of understanding of cells responses to their environment. To overcome these limitations, we focus on (1) dynamic, inducible molecular reporters and non-invasive sensors to track, and control, cell and tissue manufacturing workflows in real time, and (2) targeted modifications of intracellular pathways and cell enrichment that result in more predictable and controllable cell phenotype and function. We will begin with human mesenchymal stem cells; but the strategies can easily be applied to, e.g., to tissue specific stem cells or iPS cells. This will be done following 3 Specific Aims: Aim 1: Generating genetically encoded cell sensors for dynamic and continuous sensing of cellular phenotype and function. We will generate fluorescent and/or bioluminescent (BLI) molecular reporters specific to the tissue types in development by our CPs, based on miRNA- and promoter-driven. These reporters will emit. BLI is useful to detect gene-expression activity in early differentiation events, as quantifiable data to track these events in real-time. High-resolution fluorescence imaging allows the spatio-temporal assessment of biomarker expression at a single-cell level. TR&D-1 will collaborate with TR&Ds-2, -3 and -4 to validate their technology, providing higher throughput than commonly used end-point destructive assessments. Aim 2: Developing molecular sensors for ultra-sensitive, non-invasive monitoring of cell function. We will develop molecular sensors and reporters to sense, at very low concentration, specific biological molecules within- or produced by cells in real-time, through fluorescence and electrochemical sensing systems. Our validated universal electrochemical biosensor will be adapted to any molecule of interest using aptamers and the high specificity of the CRISPR-Cas12(13) platforms. These non-invasive measurements will be designed for integration into the Tissue Foundry automation backbone as described in TR&D-4. Aim 3: Achieving dynamic control of cell phenotype and function. After assessing the spatio-temporal dynamics of differentiation using real-time molecular reporters (SA-1 & -2), inducible genetic control and/or enrichment and pre-conditioning of cells will be implemented. This will be accomplished using inducible systems such as CRISPR-activation, Tet-inducible systems and miRNA mimicking/antagonist. Alternately cell populations can be enriched for desirable biological features through novel sorting techniques (e.g., microfluidic immunomagnetic sorting, label-free enrichment).