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.

概括 细胞实验在生物学、组织工程和药物测试研究中无处不在。然而，3D 众所周知，文化很难进行非破坏性分析。相反，通常使用牺牲来评估它们 手段：例如组织学切片，或通过粉碎样品进行化学读板机测定。这是 效率低、成本高，并且会导致数据不连续，因为每个新实验仅提供单个时间点 （如果被压碎，则进一步对整个结构进行平均）。同样，提供新的细胞或化学物质（例如， 在不干扰正在进行的实验的情况下将营养物质、药物、染料等）转移到自定义位置也很困难：仅 侵入性注射将确保到达 3D 培养物的深层。这限制了类型 可行的实验；并且，无法输送营养会导致深层细胞死亡 厚的培养物（即目前不可能将它们培养到生理相关的尺寸）。因此，有 需要能够执行流体和细胞操作（即输送、探测、移除和采样） 在活的 3D 文化中持续进行，并且对所研究的生物学影响最小。为此，广泛 拟议项目的目标是同时解决所有这些瓶颈，并另外创造一个广度 通过将 3D 培养物与自动化显微通道和端口交织在一起，实现新的实验可能性。 该想法的可行性已通过能够进行 XY 流体和 活体二维培养物中的细胞操作。因此，拟议的 R15 计划采取了接下来的逻辑步骤 通过将本发明扩展到 3D 支架（目标 1）并展示其第一个实际应用 - 连续 非破坏性时空培养分析（目标 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次 XYZ 流体/细胞操作（任务 1）。它 还设计了一种使用商业技术免提制造微流体支架的制造配方 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 支架来自 目标 1 已准备好）确定如何使用常规终点（即有毒）异位化学测定，以便 获得有关活体培养物中不同点发生的细胞行为的连续信息流。 这一目标的成功实现将能够避免对牺牲分析（例如组织学）的依赖， 这将加快实验速度，节省成本并产生连续时空生物数据的宝库。 最终，这项技术将促进基本细胞行为闭环控制的未来发展 器官大小的 3D 支架，通常将有利于涉及多个研究领域和行业 微生物培养：如生物学、再生医学、生物分子生产、药物测试、 毒理学、化妆品等。化学/生物/电子/计算机工程本科生（总共约 10 名）将接触多学科 在为期 3 年的项目过程中进行 3D 打印、微流体、显微镜和细胞培养研究。