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; 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.

概括 细胞实验无处不在生物学，组织工程和药物测试。但是，3D 众所周知，培养很难进行非破坏性分析。相反，通常使用牺牲来评估它们 方法：例如组织学切片，或通过粉碎化学板读取器测定的样品。这是 效率低下，昂贵且导致数据不连续性，因为每个新实验只提供一个时间点 （如果被压碎，则在整个构造中进一步平均）。同样，传递新的细胞或化学物质（例如， 营养素，药物，染料等）到定制位置而不打扰正在进行的实验也很困难：只有 侵入性注射将确保达到3D文化的深处。这限制了 可行的实验；而且，无法提供营养导致在深处的细胞死亡 较厚的培养物（即目前不可能将它们种植到生理相关的大小）。因此，那里 需要能够执行流体和细胞操作（即交付，探测，去除和采样） 在活着的3D培养物中，对所研究的生物学产生了最小的影响。为此，宽阔 拟议项目的目标是同时解决所有这些瓶颈，并创建一个广度 通过将3D培养物与自动微观通道和端口相交的新实验可能性。 该想法的可行性已通过能够XY流体和 活着的2D文化中的细胞操作。因此，提出的R15程序采取了下一个逻辑步骤 通过将本发明扩展到3D脚手架（AIM 1）并展示其第一个实际应用 - 连续的 无损时空培养分析（AIM 2）。具体而言，AIM 1设计一种新颖的管道建筑 能够使用最小的外部硬件（任务1）执行数千种XYZ流体/细胞操作。它 还设计了一种制造食谱，用于使用商业上的微流体脚手架进行免费制造 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脚手架 AIM 1已准备就绪）确定如何使用常规的终点（即有毒）化学测定剂以外的位置 获取有关生物文化中不同点的细胞行为的连续信息。 这个目标的成功结果将使能够避免对牺牲分析的依赖（例如，组织学）， 这将加快实验，节省连续时空生物学数据的成本和收益。 最终，这项技术将有助于未来开发基本细胞行为的闭环控制 器官尺寸的3D脚手架，通常将受益于涉及的多个研究和行业领域 微生物培养：例如生物学，再生医学，生物分子的产生，药物测试， 毒理学，化妆品等。CHEM/BIO/ELET/COMP ENG大学（总共约10个）将暴露于多学科 在3年项目过程中，3D打印，微流体，显微镜和细胞培养研究。