An Automated Microfluidics Technology for Minimally Disruptive Analysis of Cells and Fluids within Living 3D Cultures

用于对活体 3D 培养物中的细胞和液体进行最小破坏性分析的自动化微流体技术

• 批准号: 10414469
• 负责人: ROMAN S VORONOV
• 金额: $ 41.63万
• 依托单位: NEW JERSEY INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10414469
• 关键词: 3-Dimensional; 3D Print; Academic Research Enhancement Awards; Address; Adoption; Agonist; Architecture; Biological; Biological Assay; Biology; Biopsy; Blood Vessels; Cell Culture Techniques; Cell Death; Cells; Chemicals; Collagen; Collecting Cell; Complex; Computers; Controlled Environment; Cosmetics; Custom; Data; Deposition; Development; Diffusion; Drug Delivery Systems; Dyes; Ensure; Excision; Exposure to; Extracellular Matrix; Feasibility Studies; Future; Goals; Hand; Histology; In Situ; Individual; Industry; Injections; Left; Liquid substance; Literature; Location; Microfluidics; Microscopic; Microscopy; Monitor; Nutrient; Organ Size; Outcome; Pattern; Pharmaceutical Preparations; Physiological; Plumbing; Poison; Production; Reader; Recipe; Regenerative Medicine; Research; Sampling; Seeds; Specific qualifier value; Speed; Stream; System; Techniques; Technology; Thick; Time; Tissue Engineering; Tissues; Toxicology; Translating; Work; bioink; bone; cell behavior; cell type; combinatorial; cost; crosslink; design; drug testing; experience; experimental study; industry involvement; invention; microfluidic technology; microorganism; microorganism culture; multidisciplinary; new technology; novel; operation; optical imaging; osteochondral tissue; practical application; prevent; prototype; response; scaffold; scale up; spatiotemporal; success; three dimensional cell culture; undergraduate student; wasting

SUMMARY Cell experiments are ubiquitous to the studies of biology, tissue engineering and drug testing. However, 3D cultures are notoriously difficult to analyze nondestructively. Instead, they are typically evaluated using sacrificial means: such as histology sectioning, or by crushing the sample for chemical plate-reader assays. This is inefficient, costly and results in data discontinuity because each new experiment only provides a single time point (which is further averaged over the whole construct, if crushed). Likewise, delivering new cells or chemicals (e.g., nutrients, drugs, dyes, etc.) to custom locations without disturbing an on-going experiment is also difficult: only invasive injections would ensure that the deep portions of a 3D culture are reached. This limits the type of experiments that are feasible; and, the inability to deliver nutrients results in cell death in the deep portions of the thick cultures (i.e., it is currently not possible to grow them to physiologically relevant sizes). Therefore, there is a need to be able to perform fluid and cell manipulations (i.e., delivering, probing, removing, and sampling) within the living 3D cultures, continuously and with minimal effects to the studied biology. To that end, the broad goal of the proposed project is to resolve all these bottlenecks simultaneously, and additionally create a breadth of new experimental possibilities, by interlacing the 3D cultures with automated microscopic channels and ports. The feasibility of the idea has been demonstrated via a proof-of-concept prototype capable of XY fluid and cell manipulations within a living 2D culture. Therefore, the proposed R15 program takes the next logical steps by scaling up this invention to 3D scaffolds (Aim 1) and demonstrating its first practical application - a continuous nondestructive spatiotemporal culture analysis (Aim 2). Specifically, Aim 1 designs a novel plumbing architecture capable of performing thousands of XYZ fluid/cell manipulations using minimal external hardware (Task 1). It also devises a fabrication recipe for a hands-free manufacturing of the microfluidic scaffolds using commercially available 3D printers (Task 2). Simultaneously, Aim 2 uses the existing 2D prototype (until the 3D scaffold from Aim 1 is ready) to determine how to use conventional end-point (i.e., toxic) chemical assays ex-situ in order to obtain a continuous stream of information about the cell behavior occurring at different points in a living culture. A successful outcome of this aim will enable circumventing the reliance on sacrificial analysis (e.g., histology), which will speed up experiments, save costs and yield troves of continuous spatiotemporal biological data. Ultimately, this technology will facilitate the future development of closed-loop controls of basic cell behavior in organ-sized 3D scaffolds, which will generally benefit multiple fields of research and industries that involve microorganism cultures: such as biology, regenerative medicine, production of biological molecules, drug testing, toxicology, cosmetics, etc. Chem/Bio/Electr/Comp Eng undergrads (~10 total) will be exposed to multidisciplinary 3D printing, microfluidics, microscopy, and cell culturing research over the course of the 3 yr project.

总结 细胞实验在生物学、组织工程和药物检测等研究中无处不在。然而，3D 众所周知，文化很难进行非破坏性分析。相反，它们通常使用牺牲 方法：如组织切片，或通过压碎样品进行化学读板仪测定。这是 效率低、成本高，并导致数据不连续，因为每个新实验仅提供单个时间点 （如果压碎，则在整个构造上进一步平均）。同样，递送新的细胞或化学物质（例如， 营养素、药物、染料等）在不干扰正在进行的实验的情况下， 侵入性注射将确保到达3D培养的深层部分。这就限制了 实验是可行的;而且，无法提供营养物质导致细胞死亡的深层部分， 浓培养物（即，目前不可能将它们生长到生理上相关的尺寸）。因此 需要能够进行流体和细胞操作（即，输送、探测、移除和取样） 在活的3D文化中，持续地对所研究的生物学产生最小的影响。为此， 拟议项目的目标是同时解决所有这些瓶颈，并创造一个广度 新的实验可能性，通过交错的3D文化与自动显微镜通道和端口。 这个想法的可行性已经通过一个能够使用XY流体的概念验证原型得到证明， 在活的2D培养中进行细胞操作。因此，拟议的R15计划采取了以下逻辑步骤 通过将本发明按比例放大到3D支架（目标1）并展示其第一个实际应用-连续的 非破坏性时空文化分析（Aim 2）。具体来说，Aim 1设计了一种新颖的管道架构 能够使用最少的外部硬件执行数千个XYZ流体/细胞操作（任务1）。它 还设计了一种制造配方，用于商业上使用的微流体支架的免提制造， 可用的3D打印机（任务2）。同时，Aim 2使用现有的2D原型（直到3D支架从 目标1准备好了）以确定如何使用常规端点（即，毒性）化学分析，以便 获得关于活培养物中不同点发生的细胞行为的连续信息流。 这一目标的成功结果将能够避免对牺牲分析的依赖（例如，组织学）， 这将加速实验，节省成本，并产生连续时空生物数据的宝藏。 最终，这项技术将促进未来发展的基本细胞行为的闭环控制， 器官大小的3D支架，这将有利于多个研究领域和行业， 微生物培养：如生物学、再生医学、生物分子的生产、药物测试， 化学/生物/电子/比较工程本科生（共约10名）将接触多学科 3D打印，微流体，显微镜和细胞培养研究在3年的项目过程中。