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

摘要 细胞实验在生物学、组织工程和药物测试的研究中无处不在。然而，3D 众所周知，文化很难进行非破坏性分析。取而代之的是，它们通常使用牺牲性 方法：如组织切片，或通过粉碎样品进行化学平板阅读器分析。这是 效率低、成本高，并且会导致数据中断，因为每个新实验只提供一个时间点 (如果被压碎，则进一步求整个构造的平均值)。同样，输送新的细胞或化学品(例如， 营养素、药物、染料等)要在不干扰正在进行的实验的情况下定制位置也很困难：只有 侵入性注射将确保3D培养的深层部分被触及。这限制了 可行的实验；并且，不能输送营养物质会导致脑深部细胞死亡。 厚厚的培养物(即目前不可能将它们生长到与生理相关的大小)。因此，在那里 是否需要能够执行液体和细胞操作(即输送、探测、移除和采样) 在活的3D培养中，连续地，对所研究的生物学影响最小。为此，广泛的 拟议项目的目标是同时解决所有这些瓶颈，并额外创造一个广度 通过将3D培养与自动显微通道和端口交织在一起，实现了新的实验可能性。 这个想法的可行性已经通过一个概念验证原型得到了验证，该原型能够使用XY流体和 在活的2D培养中的细胞操纵。因此，拟议的R15计划采取了下一个合乎逻辑的步骤 通过将本发明放大到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(目标2)。具体地说，Aim 1设计了一种新的管道架构 能够使用最少的外部硬件执行数千次XYZ流体/单元操作(任务1)。它 还设计了一种制造配方，用于商业上使用免提制造微流控支架 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脚手架从 目标1已准备就绪)，以确定如何使用非现场常规终点(即有毒)化学分析，以便 获取有关活细胞培养中不同时间点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(例如组织学)的依赖， 这将加快实验，节省成本，并产生连续的时空生物数据宝库。 最终，这项技术将促进未来对基本细胞行为的闭环控制的发展 器官大小的3D支架，这通常将使涉及到的多个研究领域和行业受益 微生物培养：如生物学、再生医学、生物分子生产、药物检测、 毒理学、化妆品等。化学/生物/电气/化学工程专业本科生(总计约10人)将学习多学科。 3D打印、微流控、显微镜和细胞培养方面的研究。

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.