Characterization of a Droplet Microfluidic High Throughput Screening Device and Developing Machine Learning Algorithms to Study the Bone Morphogenetic Protein Signaling Pathway

项目总结 一些细胞信号系统通过混杂信号机制运行，其中多个配体可以结合 在启动导致基因表达的下游信号级联之前，与单个受体结合。 细胞中存在的混杂信号系统普遍存在于许多不同类型的生物过程中 开发和维护，到疾病，包括癌症。骨形态发生途径是一种 理想的混杂信号通路值得研究，因为在作为生长因子的10种不同的BMP配体中， 每一个都竞争性地与该途径的I型或II型受体结合。最近的研究创造了数学 骨形态发生蛋白途径中能够复制实验的混杂相互作用的模型 通过给BMP反应细胞系剂量来观察BMP途径信号转导，该细胞系在 BMP基因表达被激活，达到BMP配基滴定系列的6倍。然而，之前的结果依赖于 骨形态发生蛋白途径的矩阵组合筛选以检查反应并拟合一小部分参数 复制实验结果的数学模型。继续筛选配基组合 这样的结果导致所需的配基筛选的数量呈指数级增加。更好 需要硬件和数学工具来筛选BMP途径，以更好地理解混杂 信号现象。该项目旨在开发一种液滴微流控设备，DropShop平台， 可以以高通量的方式筛选BMP配体组合。为了做到这一点，粘附性上皮乳腺 腺体小鼠骨形态发生蛋白反应细胞系将通过一种新的微流控技术进行筛选 使用微载体的细胞培养方法。DropShop平台的液滴微流体将得到优化 研究新的细胞培养方法。在某一细胞类型中筛选BMP配体的原理证明 将在该系统中通过使用通常用于高 吞吐量液滴微流控筛选。最后，开发机器学习方法进行优化 筛选配体以减少确定BMP数学模型参数的时间，以及 帮助选择能够表征实验结果的正确模型。由此产生的系统将 展示一种液滴微流控装置的概念证明，该装置能够自动确定 混杂信号通路中的机制模型及其参数。

PROJECT SUMMARY Some cell signaling systems operate by a mechanism of promiscuous signaling, where multiple ligands can bind to a single receptor before starting a downstream cascade of signaling that results in gene expression. Promiscuous signaling systems present in cells are prevalent in many different types of biological processes from development and maintenance, to disease, including cancer. The bone morphogenetic pathway (BMP) is an ideal promiscuous signaling pathway to study because, of the 10 distinct BMP ligands that act as growth factors, each competitively binds with a type I or type II receptor of the pathway. Recent work created mathematical models of the promiscuous interactions within the BMP pathway that were able to replicate experimental observations of BMP pathway signaling by dosing a BMP-responsive cell line, which expressed YFP when the BMP gene expression was activated, to a 6-fold BMP ligand titration series. However, previous results relied on matrix combination screening of BMP pathway to examine responses and fit a small subset of the parameters of the mathematical models replicating experimental results. Continuing to screen combinations of ligands results in this manner results in an exponential increase in the number of ligand screens required. Better hardware and mathematical tools are needed to screen the BMP pathway to better understand promiscuous signaling phenomena. This project aims to develop a droplet microfluidic device, the DropShop platform, that can screen BMP ligand combinations in a high throughput manner. To do this, an adherent epithelial mammary gland murine BMP-responsive cell line will be adapted to screening by droplet microfluidics through a novel method of cell culture using microcarriers. The droplet microfluidics of the DropShop platform will be optimized to work with the novel cell culture method. Proof of principle of screening of BMP ligands in a certain cell type will be demonstrated in this system by use of a fluorescent measurement system typically used in high throughput droplet microfluidic screening. Finally, machine learning methods will be developed to optimize screening of ligands to reduce the time to determine parameter of the BMP mathematical model, as well as help in selecting the correct model that characterizes experimental results. The resulting system will demonstrate a proof of concept for a droplet microfluidic device capable of automatically determining mechanistic models and their parameters in promiscuous signaling pathways.